Lucigen 2012 Catalog Update

2012 UPDATE 

PCR & Amplification 

Cloning Kits & Vectors 

Competent Cells 

Protein Expression 

Enzymes 

Library Services

Index 

Index 

Index 

Index 

Index 

Lucigen Corporation 2012 Update 

Solutions 





Enzymes 

Custom Services 

Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

Web: www.lucigen.com 

Email: lucigen@lucigen.com 

Tech Support: 608 831 9011 

3

What’s New? 

PCR & Amplification p. 9 

• Taq98® Hot Start 2X Master Mix Kit » Unmatched ability to amplify GC-rich and 

other tough templates. Thermostability at 98°C ensures denaturation of high GC 

content targets. 

• PyroScript® RT-PCR 2X Master Mix Kit » Perform thirty-minute reverse 

transcription-PCR amplification of RNA up to 400 bp in length at >70°C without a 

separate reverse transcription step. 

• Bst DNA Polymerase, Exonuclease Minus » Highest strand displacement activity 

available for isothermal amplification and Next Generation sequencing. 

Competent Cells p. 39 

• Phage Display Electrocompetent Cells » Highest transformation efficiencies 

available (≥4 × 10 10 cfu/µg and greater). Only source for electrocompetent SS320 and 

ER2738 strains, in addition to TG1. 

• Endura Competent Cells » Direct replacement for Invitrogen Stbl3 and Agilent 

SURE strain. 

• Expanded Custom Cell Services now available 

Protein Expression p. 49 

• Expressioneering Technology » Enzyme-free directional PCR cloning in 

seconds. Complete Expresso® Systems include cloning-ready vectors and 

competent cell lines. Choice of T7 and tunable rhamnose promoters. Optional 

SUMO tags offer enhanced expression and solubility of difficult target proteins with 

6xHis-SUMO fusion tag. 

Enzymes p. 65 

• NxGen Genomic Grade Enzymes » Ideally suited to Next-Gen applications such 

as sequencing & array analysis that require effective and pure reagents. Achieve 

optimal results even in highly-sensitive applications. 

Library Services p. 79 

• NextGen Sequencing Services » Now, you can also get high-quality NextGen 

Sequencing data with fewer gaps from the same company that has successfully made 

over 1,000 libraries. We deliver unbiased multiplex BAC libraries and Roche 454 

sequencing, resulting in outstanding read length and coverage. 

4 Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 




2012 Catalog Lucigen Corporation 

Stay up to date with our 

newest products and services… 

Sign up on our web site 

www.lucigen.com to get 

the latest information about “easier, 

faster, better” tools for your research 

as soon as they are available.

What's New ......................................................................................................................... 4 

Ordering Information ...................................................................................................... 7 

1: PCR & Amplification 9 

Overview ...........................................................................................................................10 

PCR ......................................................................................................................................12 

RT-PCR ...............................................................................................................................17 

Isothermal Amplification ..............................................................................................19 

Additional Products .......................................................................................................20 

2: Cloning Kits & Vectors 23 

Overview ...........................................................................................................................24 

General Cloning ...............................................................................................................27 

PCR Cloning ......................................................................................................................30 

BAC, Fosmid, large, 

or difficult cloning ...........................................................................................................33 

Accessory Kits for Cloning ...........................................................................................35 

3: Competent Cells 39 

Overview ...........................................................................................................................40 

General Cloning & 

Library Construction ......................................................................................................42 

Custom Competent Cells .............................................................................................44 

Phage Display Library Applications ..........................................................................45 

Large or Difficult Fragment Cloning ..........................................................................46 

Index 

Index 

Index 

Index 

Index 


Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

Table of Contents 

4: Protein Expression 49 

Overview ...........................................................................................................................50 

Cloning & Expression Systems: 

Expressioneering Technology ..................................................................................51 

Competent Cells for 

Protein Expression ...........................................................................................................58 

5: Enzymes 65 

Enzymes ..............................................................................................................................66 

CAZymes ...........................................................................................................................76 

6: Library Services 79 

Overview .......................................................................................................................... 80 

Random Shear BAC/Fosmid Libraries ...................................................................... 81 

NextGen & Sanger Sequencing ................................................................................ 85 

Other Libraries/Cloning .............................................................................................. 88 

Library Screening ........................................................................................................... 92 

DNA Prep ......................................................................................................................... 94 

appendix 97 

Product Index by Product Name ................................................................................98 

Product Index by Catalog Number ...........................................................................99 

Lucigen Publications .................................................................................................... 100 

Warranty and Disclaimers .......................................................................................... 101 

Reference ........................................................................................................................ 102 

At Lucigen, we deliver advanced molecular technology, tools, and services to life scientists by inventing solutions to the most difficult problems in DNA 

cloning, amplification, and protein expression. From all of us at Lucigen, thank you for your business! 




5

Lucigen Online 

Visit lucigen.com to discover all the updates that makes it easier than ever for you to find what you 

need. Updates include: 

• Easy email sign-up! Keep up to date with the latest product introductions and promotions. 

• Online customer chat service 

• eLucidator Interactive Selection Guides: Cloning Kits & Vectors and Competent Cells 

Finding what you need has never been easier! 

Cloning: lucigen.com/cloningguide 

Competent Cells: lucigen.com/compcellguide 

Find us on Twitter & LinkedIn 

twitter.com/LucigenCorp 

linkedin.com/company/lucigen-corp. 

6 Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 




2012 Catalog Lucigen Corporation

United States customers 

On-line. www.lucigen.com. 

You may place orders on our web site using our secure Shopping Cart. 

Phone: Toll-free in the US: 888 575 9695 

Outside or within the US: 608 831 9011 

FAX: 608 831 9012 


Hours: 8 am to 5 pm Central Time, Monday-Friday, 

Distributors 

excluding most national holidays. 

International & VWR Customers: Please place orders directly with your local 

Lucigen distributor found at lucigen.com. Your local distributor prices may be 

different from the US list prices shown on this web site. Customers in countries 

without a local Lucigen distributor can place orders directly with Lucigen. Please 

contact Lucigen for more information. 

NIH Orders 

NIH researchers receive our current BPA discount by placing orders directly with 

Lucigen or through the Lucigen storefront at the NIH IntraMall: http://intramall. 

nih.gov. 

Payment 

Any major credit card can be used for payment when your order is placed. If you 

prefer to give a purchase order number, payment will be due by check or credit 

card net 30. 

Confirming Orders 

Lucigen accepts phone orders without the necessity of confirming the purchase 

order in writing. If you choose to confirm an order in writing, please clearly 

specify "CONFIRMING ORDER" to prevent duplication. Upon request, we will be 

happy to provide you with an email or fax confirmation of your order. 

High Quality and Your Satisfaction…Guaranteed! 

All Lucigen products are rigorously tested in their intended uses to ensure that 

they meet or exceed our published performance specifications. We test each 

product lot prior to stocking, and then retest on a defined schedule while that lot 

remains in stock. In addition, we monitor the performance of our products every 

day by using them in Lucigen’s new product development programs and Custom 

Services projects. Your satisfaction is our goal. If for any reason you are not satis- 

Index 

fied with a Lucigen product or service, we will gladly work with you to solve the 

problem, or to provide a replacement or credit if appropriate. 

Shipping 

All Lucigen products are shipped on dry ice by priority express delivery (10:30 

Index 

am Central time next day, unless you are in an extended service area). US orders 

received by 4:00 pm Central time will normally be shipped the same day for 

next-day delivery in the US. 

Index 

Index 

Index 


Environmentally Friendly Packaging 

Lucigen’s primary goal is to make sure you receive your order correctly without 

delay or problems. Where possible, we use recyclable material and sourcereduction 

programs to minimize environmental impact. Free Shipping 

There is no shipping charge for US orders greater than $500. For orders less than 

$500, there is a dry ice packaging and shipping charge of $35 for US deliveries. 

Free shipping is not available for international orders. 

New Lab Discounts 

If you are setting up a new lab, it’s simple to get up to $2,000 in free Lucigen 

products! See lucigen.com for more details. 

Free Sample Products 

Lucigen offers a number of free samples in PCR & Am- 

plification and Competent Cells. Limits: only one free 

sample per product per laboratory, United States and 

Canada only. Outside the United States? Please contact your local distributor. 

Large Quantity Discounts, Special Packaging & OEM 

Opportunities 

Lucigen offers substantial discounts on purchases of any of our products in large 

quantity (usually defined as 10 times more than the largest retail package size 

listed in our catalog or on the Lucigen web site). We also offer custom packaging 

and formulations of our catalog products to meet your needs. 

If your company is interested in OEM of a particular Lucigen product, please 

contact us to check availability. 

Technical Support 

Take advantage of Lucigen’s unparalleled knowledge, expertise in solving the tough- 

est cloning, expression, and amplification problems. Contact our Technical Services 

Group by phone 888 575 9695 or 608 831 9011, or techserv@lucigen.com. 

Web Site 

Lucigen’s web site at www.lucigen.com is a valuable resource for product and 

technical information. You can download PDF versions of our product manuals 

and protocols for every product we sell on the “Technical Resources” page of our 

website or the “Resources” tab on each individual product page. Poster presenta- 

tions and journal publications regarding Lucigen’s technologies are also available. 

If you are in the US or Canada, you can place orders on-line by credit card, or 

sign up to receive the Lucigen catalog and sign up to receive the latest Lucigen 

information via email. 

Technical Seminars, Technical Assistance, and Training 

Lucigen offers a free, on-site technical seminar on the latest tools and techniques 

in DNA cloning, expression, and analysis. If you are interested in having us visit 

your institution to give this seminar, please contact us at lucigen@lucigen.com or 

phone 608-831-9011. We can also arrange teleconferences between your group 

and Lucigen scientists if you would like advice and assistance with a particular 

DNA cloning or expression project. 

Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

Ordering Information 




FREE SAMPLE 

lucigen.com/samples 

7

8 Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 





Index 

Index 

Index 

Index 

Index 

SECTION 1 


Overview ..........................................10 

PCR ...................................................12 

RT-PCR .............................................17 

Isothermal Amplification ...............19 

Additional Products ........................20 


Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 




9


1 

OVERVIEW 


Lucigen offers a variety of nucleic acid amplification products designed for your needs. Currently, we offer enzymes and master mixes for PCR, reverse tran- 

scription PCR (RT-PCR), and isothermal amplification, as well as deoxynucleotides. We are continually expanding your options by discovering and developing 

novel classes of enzymes with unique capabillities. 

PCR 

NEW Taq98 Hot Start 2X Master Mix 98°C Hot-start master mix that can amplify the toughest templates while minimizing non-specific amplification. 

EconoTaq® PLUS and EconoTaq PLUS GREEN 2X Master Mixes. EconoTaq PLUS is a complete, ready-to-use PCR master mix in a ready-to-load format. 

Both mixes contain an enhancer for improved performance, and a high density buffer. Just add your DNA, primers, and water, then cycle, and load directly on 

a gel. The EconoTaq PLUS GREEN 2X Master Mix includes gel loading and tracking dyes for easy visualization. 

EconoTaq DNA Polymerase Lucigen’s EconoTaq is already established as the workhorse PCR enzyme in hundreds of labs. EconoTaq is available with a 

choice of 10X Reaction Buffer with Mg++, or without Mg++ and a separate tube of 25 mM MgCl 2 . Larger sizes are available for even greater value. 

TaqSelect DNA Polymerase Add your own dNTPs to this Taq 2X premix, formulated with an enhancer and high density buffer, to make your own master 

mix. 

PyroPhage® 3173 DNA Polymerase, Wild Type (WT) The Wild Type enzyme shows extremely high amplification fidelity due to its strong proofreading 

activity, making it useful for high-fidelity PCR. 

RT-PCR 

NEW PyroScript® RT-PCR 2X Master Mix Kit Perform one-step, thirty-minute reverse transcription-PCR to detect viral and cellular RNA. Amplify RNA up 

to 400 bp in length at >70°C without a separate reverse transcription step. 

PyroPhage® 3173 DNA Polymerase, Exonuclease Minus (Exo-) This thermostable enzyme can be used to perform both reverse transcription and PCR. 

Isothermal amplification 

NEW Bst DNA Polymerase, Exonuclease Minus (Exo-) Provides the highest strand displacement activity available for isothermal amplification and Next 

Generation sequencing. 

dNTPs 

PCR Grade dNTPs Highest quality deoxynucleotides at a great value, formulated for your convenience. Greater than 99% purity. 

10 Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 





Lucigen Amplification Enzyme Selection Guide 

Product 

Master 

Mix 

Separate 

Reaction 

Buffer(s) 

Features Applications 

dNTPs 


PCR 

Ennhancer 

Routine 

PCR 

Difficult 

PCR 

New Taq98 Hot Start 

2X Master Mix p. 12 • • • • • 

Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

Amplification 

of templates 

up to 10kb 

EconoTaq® PLUS GREEN, 

2X Master Mix p. 13 •* • • • • • 

EconoTaq PLUS, 2X 

Master Mix p. 13 • • • • • • 

EconoTaq DNA 

Polymerase p. 14 • • 

PyroPhage® 3173 DNA 

Polymerase, Wild Type 

p. 15 • • • 

New 

PyroScript® RT-PCR 2X 

Master Mix Kit p. 17 • • • • 

TaqSelect DNA 

Polymerase p. 16 • • • • 

PyroPhage® 3173 DNA 

Polymerase, Exonuclease 

Minus p. 18 • • • • 

RT- 

PCR 

Isothermal 

amplification 

New 

Bst DNA Polymerase, 

Exo Minus p. 19 • • 

*EconoTaq PLUS GREEN, 2X Master Mix, contains tracking dyes to monitor electrophoretic progress. 




11 


1


1 

Taq98 Hot Start GoTaq® Hot Start 

1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 

Platinum® PCR Supermix AmpliTaq Gold® Master Mix 

1 2 3 4 5 6 7 8 

12 Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

(75% GC genome) 

1 2 3 4 5 6 7 8 

PCR 

NEW Taq98 Hot Start 2X Master Mix 

• Unmatched ability to amplify GC-rich and other tough templates 

• Thermostability at 98°C ensures denaturation of high GC content targets 

• Hot Start Polymerase format eliminates primer dimer formation 

• Convenient 2X Master Mix 

• Great value and convenient pack sizes 

Amplify your toughest DNA templates with Taq98 Hot Start 2X Master 

Mix and minimize non-specific amplification. 

Tough PCR made simple: GC-rich templates can be hard to reliably amplify (Figure 1). 

Templates with tough structure can be just as difficult (Figure 2, C. fimi gDNA). Taq98 is 

the best option for taking control of your amplification. 

Hot Start function eliminates primer dimer formation! Robust hot-start technology 

virtually eliminates unwanted products such as primer dimers resulting from non-specif- 

ic amplification. Worry free set up reaction at room temperature. 

Extreme thermostability. The added PCR enhancer improves amplification and ther- 

mostability to give you the best PCR product possible. 

Easy to use. Taq98 2X Master Mix contains everything you need to perform your 

reaction. Simply add your template, primers and water, then cycle. 




Tough Human Templates 

Products 

Taq98 Hot Start GoTaq® Hot Start 

1 2 3 4 5 6 7 8 

1 2 3 4 5 6 7 8 

Platinum® PCR Supermix AmpliTaq Gold® Master Mix 

1 2 3 4 5 6 7 8 

ORDER INFORMATION 

1 2 3 4 5 6 7 8 

Size Cat. No. 

Taq98 Hot Start 250 rxn 30057-1 

500 rxn 30057-2 

Taq98 Hot Start is provided in a 2X Master Mix format. All required 

buffer components, enzyme, Mg++ and dNTP’s are all present in optimized 

concentrations. Only template DNA, primers and nuclease-free water are 

required to set up the reaction. Please see the user manual for composition 

information. Standard reaction size is 50 µL. 


FREE SAMPLE 


Lane %GC 

1 78.0% 

2 76.4% 

3 75.2% 

4 70.6% 

5 68.6% 

6 64.6% 

7 59.5% 

8 56.7%

PCR 

EconoTaq® PLUS and EconoTaq PLUS GREEN 2X Master Mixes 

• Outstanding performance & value make them perfect for routine PCR. 

• Ready-to-use PCR reaction master mixes contain 

dNTPs & PCR Enhancer. 

• EconoTaq PLUS GREEN contains tracking dyes. 

• Can be cycled up to 98°C to amplify challenging GC-rich templates 

others cannot. 

Performance, Convenience, Reliability, and Value for Routine PCR. 

More effective than other Taq master mixes. EconoTaq PLUS and PLUS GREEN 2X Master Mixes contain 

a proprietary PCR enhancer, so they can successfully amplify templates that fail to amplify with other 

PCR master mixes (Figure 1). High density reaction buffer enables direct gel loading following PCR. 

Excellent results in routine PCR. EconoTaq PLUS GREEN & EconoTaq PLUS contain high purity, high 

activity EconoTaq DNA Polymerase for reliable amplification of templates up to 5 kb 

(Figure 1). 

Amplify challenging GC-rich templates. These 2X Master Mixes can be cycled up to 98°C to amplify 

challenging GC-rich templates others cannot (Figure 2). 

Convenient tracking dyes in EconoTaq PLUS GREEN. On a 1% agarose gel, the blue dye migrates at 

the same rate as a 5 kb DNA fragment, and the yellow dye migrates at 75 bp. See Figure 3. If desired, 

the dyes are easily removed by standard DNA purification products or ethanol precipitation. For 

fluorescence or absorbance detection assays, EconoTaq PLUS 2X Master Mix offers the same great 

performance without the dyes. 

Flexible & easy to use. EconoTaq PLUS GREEN and EconoTaq PLUS are packaged in 50 reaction vials 

that can be stored in the refrigerator (+4ºC) for up to 3 months. They are stable for at least 10 freeze- 

thaw cycles. 

Products 

Size Cat. No. 

EconoTaq PLUS GREEN 2X Master Mix 10 reactions 30033-0 


Use EconoTaq ® PLUS GREEN 2X Master Mix for PCR in which the products will be analyzed by 

agarose gel electrophoresis and ethidium bromide staining. Use EconoTaq PLUS for PCR in which the products 

will be analyzed by absorbance or fluorescence excitation, to avoid possible interference by the presence of the 

dyes. One free sample per lab. See page 7 for additional information. 


FREE SAMPLE 


500 reactions 30033-1 

1,000 reactions 30033-2 

EconoTaq PLUS 2X Master Mix 50 reactions 30035-0 


1,000 reactions 30035-2 

Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

Figure 1. EconoTaq PLUS GREEN Master Mix vs. GoTaq® 

Green master mix (Promega) in PCR. PCR was performed 

according to the manufacturer’s recommendations using 

primers specific for the indicated genes. Panel A, 0.7 

kb prokaryotic single-stranded DNA binding protein 

(genomic DNA); Panel B, 2.7 kb prokaryotic DNA polymerase 

A (genomic DNA); Panel C, 4.5 kb gene (plasmid 

DNA). 

2 kb� 

1.5 kb� 

1 kb� 

0.7 kb� 

0.5 kb� 

MW 

PLUS GREEN 

A B C 

GoTaq 

PLUS GREEN 

GoTaq 

5 kb 

3 kb 

2 kb 

1 kb 

Figure 3. Easy visualization with EconoTaq PLUS GREEN. 

During electrophoresis, the green loading color separates 

into a slow-moving blue dye and a fast-moving 

yellow dye. 

PLUS GREEN 

GoTaq 

MW 

EconoTaq PLUS 

MW 75% 63% 65% 58% 60% 60% 

Invitrogen 

MW 75% 63% 65% 58% 60% 60% 

2 kb� 

1.5 kb� 

1 kb� 

0.7 kb� 

0.5 kb� 

NEB 

MW 75% 63% 65% 58% 60% 60% 

2 kb� 

1.5 kb� 

1 kb� 

0.7 kb� 

0.5 kb� 

Figure 2. Figure 2. GC-rich regions from human 

genomic DNA were PCR amplified using 98° C 

denaturation. 




13 


1

No DNA 

34 No DNA 


1 

Buffer) DNA 

132 133 134No Shh 

Cdo wt 

Cdo mut 

Gas1 mut 

Promega Lucigen NEB 

MW – + – + – + – + MW 

Figure 4. Taq DNA polymerase from Promega 

and New England Biolabs were compared to 

Lucigen’s EconoTaq DNA Polymerase (2 different 

lots) in amplifying the ampicillin gene (0.8 kb) in 

a pUC19 vector. (–), no DNA. (+), DNA added 

(40 ng). MW, 1 kb ladder. 

MW Std. 

MW Std. 

Hip1 (AmpliTaq + 10X GSB) 

124 125 126 127 128 129 130 131 132 133 134 

Hip1 (EconoTaq + 10X EconoTaq Buffer) 

124 125 126 127 128 129 130 131 132 133 134 

AmpliTaq EconoTaq 

Figure 5. EconoTaq vs. AmpliTaq® (Applied Biosystems) 

DNA polymerase in genotyping. All PCR reactions were 

performed in a RoboCycler 96 (Stratagene). Hip1 genotyping 

was performed using the following PCR conditions: 

94°C for 1min, 35 cycles of 94°C for 30sec, 62°C 

for 60sec, 72°C for 90sec, and 72°C for 7min. Shh, Cdo 

and Gas1 genotyping were performed using the following 

PCR conditions: 94°C for 2min, 35 cycles of 94°C for 

60sec, 65°C for 60sec, 72°C for 90sec, and 72°C for 7min. 

All PCR reactions contained final concentrations of 1 µM 

of each primer, 200 µM dNTPs, 1X cresol red loading dye, 

and 1U of the indicated Taq polymerase. Sequences for 

all PCR primers have been previously published (references 

available). Data courtesy of Dr. Benjamin Allen, 

Dept. Molecular & Cellular Biology, Harvard University. 

14 Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

No DNA 

No DNA 

Shh 

Cdo wt 

Cdo mut 

Gas1 mut 

PCR 

EconoTaq® DNA Polymerase 

• Great Taq Polymerase, performance at a low price. 

• Choice of reaction buffers, with or without MgCl 2 . 

QC specifications for EconoTaq are rigorous: 

• Greater than 99% pure by SDS gel electrophoresis. 

• No detectable DNA contamination as determined by PCR using generic primers. 

• No detectable endonuclease (nicking) activity. Incubation of 10 U of EconoTaq DNA Polymerase 

AmpliTaq EconoTaq 

with 1 µg of supercoiled pBR322 DNA for 16 hours at 70°C results in no detectable conversion to 

relaxed or linear forms by agarose gel electrophoresis. 

• No detectable exonuclease activity. Incubation of 10 U of EconoTaq DNA Polymerase with 1 µg of 

Hind III-cut lambda DNA for 16 hours at 70°C results in no smearing of bands on agarose gels. 

EconoTaq Performance 

As shown in Figures 4 and 5 Lucigen’s EconoTaq DNA Polymerase performs as well as, or better than, 

more expensive Taq preparations from several other suppliers in routine PCR. 

Products 






FREE SAMPLE 


At $89 for 1000 units list price (quantity discounts available), EconoTaq’s low price is coupled with high 

quality and performance. 

Size Cat. No. 

EconoTaq DNA Polymerase (with Mg ++ ) 250 U 30031-0 

1,000 U 30031-1 

5,000 U 

(5 × 1,000 U) 

10,000 U 

(10 × 1,000 U) 

30031-2 

30031-3 

EconoTaq DNA Polymerase (separate Mg ++ ) 1,000 U 30032-1 

5,000 U 

(5 × 1,000 U) 

10,000 U 

(10 × 1,000 U) 

30032-2 

30032-3 

EconoTaq DNA Polymerase is provided with a choice of 10X Reaction Buffer with Mg ++ (“with Mg ++ ”); or 

10X Reaction Buffer without Mg ++ and a separate tube of 25 mM MgCl 2 (“separate Mg ++ ”). 

Please inquire for bulk pricing. One free sample per lab. See page 7 for additional information.

PCR 

PyroPhage® 3173 DNA Polymerase, Wild Type 

• Strong proofreading activity. 

• Can be thermocycled for PCR. 

Superior DNA amplification with the first thermostable phage DNA polymerase 

Lucigen’s PyroPhage 3173 DNA Polymerase (patent pending) is the first thermostable enzyme to offer 

the superior DNA replication performance of a phage replicase. 

The thermostability of PyroPhage 3173 DNA Polymerase makes it useful for thermocycling methods, 

including PCR. The potent 3’-5’ exonuclease (proofreading) activity of the Wild Type enzyme results 

in extraordinarily high fidelity. 

Advantages 

Superior PCR amplification 

PyroPhage 3173 DNA Polymerase is much more effective than current PCR enzymes in amplifying 

certain difficult templates. PyroPhage 3173 DNA Polymerase effectively amplifies most templates of up 

to 4 kb. 

Superior replication fidelity. 

PyroPhage 3173 DNA Polymerase (Wild Type) demonstrates extremely strong proofreading activity 

and copy fidelity (Figure 7). Because of this very strong exonuclease activity, use of phosphorothioated 

primers is recommended when using the Wild Type enzyme for DNA amplification. 

Usage Information 

As with most PCRs, amplification is highly dependent on primers and targets. Particular primer/ 

template sets can result in undesired reaction products. Such variability may be reduced by the use 

of alternative primer sets. Cycling conditions should be optimized using conditions under which the 

PyroPhage 3173 DNA Polymerase performs best. Increasing annealing temperatures during PCR 

improves the reaction specificity of PyroPhage 3173 DNA Polymerase. Cold reaction set-up and use 

of 2 step PCR cycling parameters with a combined annealing/extension step at a temperature of about 

72°C greatly shortens cycling time and is recommended for best results. 

Legal Information 

The PyroPhage DNA Polymerases and methods of using the PyroPhage or support the unauthorized or unlicensed 

use of the PCR process, reverse transcription PCR process or isothermal DNA synthesis. It is the sole responsibility of 

the buyer to ensure that use of the product does not infringe the patent rights of third parties. 

Products 

Size Cat. No. 

PyroPhage 3173 DNA Polymerase, Wild Type 500 U 30051-1 

2,500 U 30051-2 

5,000 U 30051-3 

PyroPhage 3173 2X PCR Buffer, 500 rxns 12.5 ml 30072-1 


PyroPhage 3173 DNA Polymerase, Wild Type is provided with PyroPhage 3173 2X PCR Buffer, and PCR 

Control + Primers. 2X PCR Buffer is also available separately. The 2,500 U and 5,000 U package sizes of 

PyroPhage 3173 DNA Polymerase are provided as multiples of the 500 U package size. 


9 x 10 4 

7 x 10 4 

5 x 10 4 

3 x 10 4 

1 x 10 4 

Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

0 

PyroPhage 

Wild Type 

PyroPhage 

Exo Minus 

Fidelity of PCR Enzymes 

Taq 

Vent 

Figure 7. PyroPhage 3173 fidelity. Fidelity measurements, 

shown as the ratio of correct to incorrect nucleotide 

incorporations, are based on the LacIq forward 

mutation assay (1, 2). PyroPhage 3173 DNA Polymerase 

(Wild Type and Exo Minus) was compared to various 

commercially available enzymes. 

KOD 

Phusion 




PlatTaqHF 

15 


1


1 

Stay up to date with our 

newest products and services… 

Sign up on our web site 

www.lucigen.com to get 

the latest information about “easier, 

faster, better” tools for your research 

as soon as they are available. 

16 Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

PCR 

TaqSelect DNA Polymerase 

• 2X Taq Polymerase pre-mix with PCR enhancer. 

• Optimize with your own dNTPs, available separately. 

Outstanding PCR performance 

Lucigen offers its high quality Taq polymerase in a 2X Premix containing a stabilizer/enhancer opti- 

mized for performance. The high density reaction buffer contains magnesium at an optimal concentra- 

tion for PCR and allows direct loading after thermocycling. TaqSelect DNA Polymerase offers the same 

high performance of EconoTaq PLUS 2X Master Mix, but does not contain dNTPs (deoxynucleotides). 

Applications 

• Routine PCR. 

• Genotyping. 

• Amplify challenging templates. 

• Preparation of PCR fragments for cloning. 

Products 


TaqSelect DNA Polymerase is formulated as a 2X Premix. The standard reaction size is 50 µl. One free sample 

per lab. See page 7 for additional information. 





FREE SAMPLE 


Size Cat. No. 

TaqSelect DNA Polymerase 10 reactions 30041-0 


1,000 reactions 30041-2

RT-PCR 

NEW PyroScript ® RT-PCR 2X Master Mix Kit 

• Fast - Amplify viral and cellular RNA in 30 minutes! 

• High Temperature - Reverse Transcription at > 70°C. 

• Convenient - Just add primers and RNA template. 

• Effective - Real-time or end-point analyses. 

The PyroScript RT-PCR 2X Master Mix Kit incorporates a thermostable PCR enzyme discovered by 

Lucigen that also has efficient reverse transcription activity. PyroScript RT-PCR 2X Master Mix simplifies 

RNA detection by allowing accurate single-tube, single-enzyme reverse transcription PCR (RT-PCR) of 

amplicons up to 400 bp. This format reduces hands-on time and facilitates error-free reaction set up. Just 

add template, primers and cycle. Detect amplification by gel electrophoresis or real-time analysis. 

Low background fluorescence and enzyme compatibility with commonly used fluorescent stains make 

PyroScript RT-PCR 2X Master Mix excellent for qRT-PCR. PyroScript RT-PCR 2X Master Mix is especially 

useful for RT-PCR detection and quantification of viral as well as transcript RNA. Extreme thermostability 

allows denaturation of crude specimens at 94°C to simplify sample preparation. Reverse transcription at 

70°C or even higher improves analysis of highly-structured RNA templates. 

Amplify up to 400 bp of transcript RNA and viral RNA 

PyroScript RT-PCR 2X Master Mix was used to detect Human 18S rRNA sequences and viral RNA, as 

shown in figure 8. 

PyroScript RT-PCR 2X Master Mix is Sensitive 

The sensitivity of PyroScript RT-PCR 2X Master Mix is shown in figure 9, in which as few as 1.2 copies of 

MS2 bacteriophage RNA were detected. 

Linear RT-PCR Quantification with PyroScript RT-PCR 2X Master Mix 

PyroScript RT-PCR 2X Master Mix was used in real time analysis, as shown in figure 10, to illustrate the 

linearity of quantification. 


The methods of using the PyroScript RT-PCR 2X Master Mix Kit are covered by pending patents assigned to Lucigen 

Corporation. Lucigen does not encourage or support the unauthorized or unlicensed use of the PCR process, reverse 

transcription PCR process or isothermal DNA synthesis. It is the sole responsibility of the buyer to ensure that use of the 

product does not infringe the patent rights of third parties. 

Products 

PyroScript® RT-PCR 2X 

Master Mix Kit 

Ordering Information 

Size Cat. No. 

50 rxn 

(Trial Size) 

PyroScript RT-PCR 2X Master Mix Kit is supplied in vials filled with 50 reactions per vial. The master mix is 

stable for at least 10 freeze-thaw cycles. Also supplied is an extractable RNA control, Control primer set, 

100 mM Magnesium sulfate, and Nuclease-free water. 


30055-0 

100 rxn 30055-1 

500 rxn 

(5 x 100 rxn) 

30055-2 

RFU 

5,000 

0 2,500 

Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

A. Human 18S rRNA B. Viral RNA 

100 bp 87 244 367 262 249 

Ladder bp bp bp bp bp 

%GC 51% 56% 54% 65% 53% 

100 bp 

Ladder 

100 bp 89 124 93 160 217 218 362 243 294 

Ladder bp bp bp bp bp bp bp bp bp 

Figure 8. Single Enzyme RT-PCR A. Human 18S rRNA 

sequences were amplified from 100 pg total A549 

cell line RNA. Five primer sets targeting amplicons of 

between 51 and 65% GC content and 87 to 367 bp in 

length were tested. B. Viral RNA (Enterobacteriophage 

MS2, ATCC 15597-B1) was amplified by 40 cycles of 

RT-PCR without background. Products from 89 to 362 

bp in length were amplified. One-step single-enzyme 

RT-PCR cycle conditions: 15 sec @ 94°C (10s @ 94°C, 

30s @ 72°C)*40. 

12×10 4 12×10 3 12×10 2 12×10 1 12 1.2 NTC 

COPIES 

Ampli�cation Curves 

10 15 20 25 30 35 40 




100 bp 

Ladder 

Figure 9. Single-enzyme, one-step RT-PCR from a 

10-fold dilution series of quantitated MS2 bacteriophage 

RNA. 

Pyroscript RT-PCR Master Mix amplified RNA copy 

numbers from 120,000 to 1.2 copies (as estimated by 

OD 260 ). No amplification was detected in the no target 

control (NTC). 

Figure 10. Quantitative RT-PCR 

Triplicate single-enzyme RT-PCR amplifications of a 10 3 

to 10 8 -fold dilution series of the RNA target. Amplification 

was detected by EvaGreen fluorescence. PyroScript 

RT-PCR 2X Master Mix, RNA Control and Control Primer 

Set were used for the analysis. 

17 


1


1 

1 2 

Actin 

Figure 11. Single-tube, single-enzyme RT-PCR with 

PyroPhage 3173 DNA Polymerase (Exo Minus). 

Mouse liver RNA was used as a template with actinspecific 

primers. A single band was produced at the 

expected size of 283 bp. 

18 Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

RT-PCR 

PyroPhage® 3173 DNA Polymerase, Exonuclease Minus (Exo-) 

Superior DNA amplification 

The thermostability of PyroPhage 3173 DNA Polymerase makes it useful for thermocycling methods, 

including PCR. PyroPhage 3173 DNA Polymerase also has an efficient reverse transcription activity. 

The enzyme can perform single-tube, single enzyme reverse-transcription PCR of RNA transcripts. 

The Exo Minus mutant lacks the potent 3’-5’ exonuclease (proofreading) activity of the Wild Type 

enzyme. 

RT-PCR capabilities. 

The PyroPhage 3173 DNA Polymerase Exo-enzyme can be used for single-tube, single-enzyme 

reverse transcription PCR (RT-PCR), as shown in Figure 11. 

Usage Information 

As with most PCRs, amplification is highly dependent on primers and targets. Particular primer/ 

template sets can result in undesired reaction products. Such variability may be reduced by the use 

of alternative primer sets. Cycling conditions should be optimized using conditions under which the 

PyroPhage 3173 DNA Polymerase performs best. Eliminating the low temperature RT incubation step 

prior to denaturation for RT-PCR, and increasing annealing temperatures during PCR improves the 

reaction specificity of PyroPhage 3173 DNA Polymerase. Cold reaction set-up and use of 2 step PCR 

cycling parameters with a combined annealing/extension step at a temperature of about 72°C greatly 

shortens cycling time and is recommended for best results. 


The PyroPhage DNA Polymerases and methods of using the PyroPhage DNA Polymerases are covered by pending 

patents assigned to Lucigen Corporation. Lucigen does not encourage or support the unauthorized or unlicensed 

use of the PCR process, reverse transcription PCR process or isothermal DNA synthesis. It is the sole responsibility of 

the buyer to ensure that use of the product does not infringe the patent rights of third parties. 

Products 


PyroPhage 3173 DNA Polymerase, Exo Minus is provided with PyroPhage 3173 2X PCR Buffer, and PCR 

Control + Primers. 2X PCR Buffer is also available separately. The 2,500 U and 5,000 U package sizes of 

PyroPhage 3173 DNA Polymerase are provided as multiples of the 500 U package size. 





Size Cat. No. 

PyroPhage 3173 DNA Polymerase, Wild Type 500 U 30053-1 

2,500 U 30053-2 

5,000 U 30053-3 

PyroPhage 3173 2X PCR Buffer, 500 rxns 12.5 ml 30072-1

ISOTHERMAL AMPLIFICATION 

NEW Bst DNA Polymerase, Exonuclease Minus 

• Highest strand displacement activity available. 

• Used in isothermal amplification and Next Generation Sequencing. 

• High specific activity for DNA amplification and sequencing. 

Applications 

• Strand displacement amplification 

• DNA sequencing through GC rich regions 

• Rapid sequencing from nanogram amounts of DNA template 

Bst DNA Polymerase, Exonuclease Minus, is a recombinant form of the 67 kDa Bacillus stearothermophi- 

lus DNA Polymerase protein (large fragment). The enzyme has 5’-3’ polymerase activity and strand 

displacement activity, but it lacks 3’-5’ exonuclease activity. It also has reverse transcription activity. 

Lucigen's Bst DNA Polymerase, Exonuclease Minus, has higher strand displacement activity than that 

of other suppliers (Figure 12). The enzyme can be used in nucleic acid amplification methods such as 

isothermal amplification, whole genome amplification (WGA), and multiple displacement amplification 

(MDA). It also can be used in Next Generation sequencing. 

This enzyme has optimal activity at 65°C. It is suitable for sequencing DNA with high GC content and 

secondary structures. It is available in concentrations of 8,000 U/ml or 50,000 U/ml. 

Tips for use: 

• Requires 0.1% Triton X-100 for long term storage. 

• Reaction temperatures above 70°C are not recommended. 

• Bst DNA Polymerase cannot be used for thermal cycle sequencing. 

Heat Inactivation: 80°C for 20 min. 


Some uses for this product may require licenses. Lucigen does not encourage or support the unauthorized or unlicensed 

use of patented nucleic acid amplification processes for isothermal amplification, whole genome amplification 

(WGA), multiple displacement amplification (MDA), and Next Generation sequencing. It is the sole responsibility of the 

buyer to ensure that use of the product does not infringe the patent rights of third parties. If the purchaser is not willing 

to accept these use limitations, Lucigen Corporation is willing to accept return of the product for a full refund. 

Products 

Bst DNA Polymerase, Exonuclease Minus 

8,000 U/ml 

Bst DNA Polymerase, Exonuclease Minus 

50,000 U/ml 


Size Cat. No. 

200 U 30027-0 

2,000 U 30027-1 

10,000 U 

(5 × 2,000 U) 

30027-2 

10,000 U 30028-1 

Bst DNA Polymerase, Exonuclease Minus is available in two concentrations, 8,000 U/ml and 50,000 U/ml, in 

a storage buffer of 10 mM Tris-HCl, pH 7.5, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.1% Triton X-100, and 

50% Glycerol. Also supplied is 10 X DNA Polymerase Buffer B, composed of 200 mM Tris-HCl pH 8.8, 100 

mM (NH 4 ) 2 SO 4 , 100 mM KCl, 20 mM MgSO 4 , and 1.0 % Triton X-100. One free sample per lab. See page 7 

for additional information. 


FREE SAMPLE 


Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

Supplier E Lucigen Supplier N 

Bst + + - + + - + + - 

Primer + - - + - - + - - 

MW 




�10 kb 

�3 kb 

�1 kb 

Figure 12. Lucigen Bst DNA Polymerase, 

Exonuclease Minus, possesses greater strand- 

displacing polymerase activity. 

M13 single stranded DNA was incubated with or without 

8 units of Bst DNA Polymerase(+/- Bst) in reaction 

buffer supplied by the manufacturer, with or without 

replication primer (+/- primer) for 30 minutes at 65°C 

MW, 1 kb ladder. 

19 


1


1 

20 Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

ADDITIONAL PRODUCTS 

PCR Grade dNTPs 

• Convenient formulations. 

• Highest quality: > 99% purity. 

• Tested in PCR. 

• Great value! 

Highest quality deoxynucleotides, 2'-deoxynucleoside 5'-triphosphates, 

at a great value, formulated for your convenience. 

These nucleotides are also suitable for: 

Sequencing Nick translation cDNA synthesis 

TdT-tailing reactions Fill-in reactions 

2.5 mM dNTP Mix, PCR Grade 

This ready-to-use dNTP Mix is a mixture of all four nucleotides (dATP, dCTP, dGTP, dTTP), each 

dissolved in highly purified water at a concentration of 2.5 mM, at pH 8.3. Two milliliters of the Mix is 

enough for 500 standard 50 µl EconoTaq® or TaqSelect DNA Polymerase PCR reactions. Free sample 

available, see lucigen.com. 

10 mM dNTP Set, PCR Grade 

This high-purity dNTP set allows you to formulate your own PCR mix. Each of all four nucleotides (dATP, 

dCTP, dGTP, dTTP) is provided in a separate vial (750 µl each), dissolved at a concentration of 10 mM in 

highly purified water, pH 8.3. 

Products 


The 2.5 mM dNTP Mix is a mixture of all four nucleotides, each dissolved in highly purified water at a 

concentration of 2.5 mM, at pH 8.3. The 10 mM dNTP Set provides each nucleotide in a separate vial 

(750 µl each), dissolved at a concentration of 10 mM in highly purified water, pH 8.3. One free sample per 

lab. See page 7 for additional information. 





Size Cat. No. 

2.5 mM dNTP Mix, PCR Grade 2 ml (2 × 1ml) 30030-1 

10 mM dNTP Set, PCR Grade 

10 ml 

(5 × Cat. No. 30030-1) 

20 ml 

(10 × Cat. No. 30030-1) 

750 μl of each dNTP 

5 × Cat. No. 30029-1 

30030-2 

30030-3 

30029-1 

30029-2

SECTION 2 


Overview ..........................................24 

General Cloning ...............................27 

PCR Cloning .....................................30 

BAC, Fosmid, large, 

or difficult cloning ...........................33 

Accessory Kits for Cloning .............35 


Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

Solutions 




23


2 


Lucigen has developed a wide variety of cloning systems to capture nearly 

any type of insert, from routine fragments to the “unclonable”. Novel tran- 

scription-free vectors reduce cloning bias, improve efficiency, and increase 

throughput. Most vectors are pre-cut, dephosphorylated, and ready-to-use. 

Kits are available for general cloning, PCR cloning, and for BAC/Fosmids 

cloning of large inserts, and difficult DNAs. Most kits are available with 

competent cells. Lucigen also provides kits for DNA end-repair and ligation, 

as well as DNA ladders and specialty vectors. 

General Cloning 

CloneSmart® Cloning Kits (pSMART® Vectors) » Stably clone any insert 

in transcription-free, blunt vectors. Choice of antibiotic resistance and copy 

number. 

pEZ Seq Cloning Kits » Clone most inserts into blunt vectors. Option for 

blue/white screening. 

PCR Cloning 

GC Cloning and Amplification (pSMART® GC Vectors) » Stably clone 

PCR products from proofreading or non-proofreading enzymes. 

GC Cloning and Amplification (pGC Blue Vector) » Clone PCR prod- 

ucts with optional blue/white screening; includes T7 & SP6 RNA polymerase 

promoters. 

BigEasy® Long PCR Cloning Kit (pJAZZ® Vectors) » Clone any amplifica- 

tion product, even “unclonable” fragments, up to 30 kb in pJAZZ® Vectors 

configured for PCR cloning. 

BAC, Fosmid, Large, or Difficult Cloning 

CopyRight®: v2.0 BAC Cloning Kits (pSMART® BAC and pEZ BAC) » 

Efficiently clone any insert up to 200kb. 

CopyRight®: v2.0 Fosmid Cloning Kits (pSMART®FOS) » Efficiently 

clone any insert 35-45kb. 

BigEasy® v2.0 Linear Cloning System (pJAZZ® Vectors) » Clone any 

insert up to 30kb, even A/T-rich, G/C-rich, or repetitive DNA, in the unique 

pJAZZ® linear vector. 

For a full list of our specialty vectors, visit lucigen.com. 

Table 1. Comparison of pSMART versus conventional cloning vectors 

pSMART 

Vectors 

Conventional 

Vectors 

Screening 

Method 

24 Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

Toxic 

ORFs 

AT-Rich/GC- 

Rich DNA 

Strong 

Promoters 




Accessory Kits for Cloning 

Lucigen offers a wide range of DNA & PCR end repair kits, ligation, and 

adapter fill-in kits, and DNA ladders. 

Transcription-Free Cloning Vectors: 

A Powerful Tool for Trouble-free Cloning 

Lucigen’s CloneSmart® Technology utilizes transcription- and translation- 

free cloning vectors that eliminate potential difficulties with cloning DNA. 

Sequences that are difficult or impossible to clone in conventional vectors 

are efficiently obtained in Lucigen’s patented pSMART® series of vectors 

(Table 1). 

Disadvantages of conventional vectors 

Most conventional vectors are based on the plasmid pUC19. While pUC 

vectors are effective for cloning many inserts, transcription of insert se- 

quences from the lacZ promoter may select against recombinant plasmids, 

particularly those containing toxic coding regions, short repeats, AT- or 

GC-rich regions, or strong secondary structures. Strong promoters are 

likewise difficult to clone in pUC. Blue/white colony screening can introduce 

colony-picking errors (false positives and false negatives) that further skew 

clone representation. High copy number or ampicillin selection can cause 

instability of certain clones as well. These traits result in biased libraries, lost 

clones, sequencing gaps, or other undesirable outcomes that increase the 

effort to find a particular clone or finish a sequence assembly. 

Advantages of pSMART Vectors 

The pSMART vectors are designed to prevent transcription into or out of 

the cloned insert DNA. Vector-driven transcription into the insert has been 

eliminated by removal of the lacZ promoter and the lacZα gene. Transcrip- 

tion terminators flank the cloning site to prevent cloned promoters from 

interfering with plasmid selection and replication functions. In addition, a 

transcription terminator placed downstream of the drug resistance gene 

prevents read-through of insert sequences from this promoter. The pSMART 

vectors are available in four versions: Kanamycin or Ampicillin resistant, and 

High Copy or Low Copy origins of replication. Cloning efficiencies with the 

pSMART vectors are generally >99%, so colony screening is not needed. The 

pSMART-cDNA vector (see lucigen.com) also contains the T7 promoter for 

in vitro transcription/translation. If a ribosomal binding site and ATG codon 

are included in the insert, this vector can be used for in vivo expression in 

Lucigen’s E. cloni® EXPRESS BL21(DE3) Cells or OverExpress C41(DE3) 

and C43(DE3) Cells. The pSMART BAC vector (CopyRight Kits) includes 

additional structural features to optimize cloning of very large fragments (≥ 

150 kb). 

Secondary Structures 

(e.g., repeats or hairpins) 


Large 

Inserts 

Short ORFs, 

Weak Promoters 

None 

Required ✓ ✓ ✓ ✓ ✓ ✓ 

Blue/White � � � � � 

? 

(false negatives 

common)

Finding the right vector has never been easier! 

Table 2. Selected applications for Lucigen cloning kits Highlighted: Best kit for the application 

Select specific insert characteristics, preferred vector features, or both 

A 

to instantly see relevant results, sorted by recommendation or features. 

Fosmids cDNAs Expression 

BACs 

(< 200 kb) 

“Unclonable” 

AT-Rich, 

Repeats, 

(0-30 kb) 

General 

Cloning, 

PCR Products, 

Toxic ORFs 

(5-10 kb) 

General 

Cloning 

(


2 

Table 3. Selected Vectors for Cloning 

Vector Applications Insert Size Insert Site Benefits Level of Difficulty 

BigEasy ® v2.0 Linear Cloning 

Systems 

telN repA 

telN rep e A 

telN rep e A 

ROP 

Inc C 

Ori 

Ori 

NotI 

AhdI 

ApaI 

(SmaI) 

26 Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

cB 

cB 

cB 

pSMART ® 

GC HK 

1862 bp 

Kan r 

ori 2 

pSMART VC 

6.8 kb 

pSMART ® 

BAC 

7.5 kb 

cosN 

Terminator 

Kan r 

rep E par A par B par C 

Ori 

Kan r 

ori V 

SL1 primer 

SR4 primer 

Cmr 

pSMART ® -cDNA 

1.8 kb 

pSMART ® GC LK 

2054 bp 

NotI 

AhdI 

PmlI 

EcoRI 

HindIII 

BamHI 

BstXI 

c c 

sacB lacZ 

BstXI 

BamHI 

HindIII 

EcoRI 

PmlI 

AhdI 

NotI 

(SmaI) 

AhdI 

NotI 

SL1 

primer 

SR2 

primer 

Cam r 

Kan r 

Kan r 

Stuffer 3.3Kb 

CL3 

primer 

SR2 

primer 

C 

SL1 

primer 

SR2 

primer 

EcoRV 

EcoRI 

C 

EcoRI 

XbaI 

EcoRV 

SwaI 

C 

C 

pJAZZ-OC 

pJAZZ-OK 

pJAZZ-OK GC 

T7 

EcoRV 

SalI/HincII 

EcoRI 

BamHI 

Blunt 

Blunt 

BamHI 

EcoRI 

NotI 

XhoI 

EcoRV 

SwaI 

EcoRV 

EcoRI 

EcoRI 

XbaI 

EcoRV 

SwaI 

Very difficult DNA 

Large inserts 

Long PCR roducts 

BAC/Large 

insert cloning 

Up to 30 kb 

Up to 300 

kb 




NotI 

Blunt 

C-overhang 

BamHI 

Blunt 

Problematic DNA Up to 12 kb Blunt 

cDNA libraries Up to 10 kb 

Problematic 

PCR products 

(from any PCR 

polymerase) 

General PCR 

products 

(from any PCR 

polymerase) 

General cloning 

and small 

problematic DNA 

Up to 15 kb 

Up to 10 kb 

Up to 10 kb Blunt 

EcoRI/NotI 

NotI/Blunt 

Blunt/Blunt 

C-overhang 


Highest fidelity cloning system known 

Stably clone any DNA 

High fidelity cloning 

Clone at single copy; induce multiple 

copies of insert for DNA preparation 

Insert stability assured due to single 

copy cloning 

Stably maintains problematic DNA 

at 20–50 copies/cell 

Clone: 

Toxic genes 

Strong promoters 

AT- or GC-rich DNAs 

Much more accurate cDNA libraries 

and microarrays 

No cloning bias 

High insert stability 

More accurate than TOPO ® or TA ® 

cloning 

Greater insert stability 

No colony screening 

No lost clones 

Includes enzymes & reagents for PCR, 

G-tailing and ligation, and cells 

High-fidelity cloning 

Greater insert stability 

Wide insert range 

No colony screening 

No lost clones 

Most 

problematic 

DNA 

Least 

problematic

GENERAL CLONING 

CloneSmart® Cloning Kits (pSMART® Vectors) 

• Convenient, pre-processed vectors eliminate the need for 

digestion, gel purification, and dephosphorylation. 

• Transcription terminators flanking the cloning site stabilize otherwise 

unclonable inserts. 

• Choice of copy number and antibiotic resistance. 

• Available with a choice of competent cell transformation efficiencies. 

Clone quickly and easy manipulation 

Lucigen’s unique cloning kits with pre-processed vectors and highly competent cells eliminate hours of 

tedious preparation and enable efficient cloning. Ligation can be completed in as little as 30 minutes. 

No clean-up needed; only a brief heat denaturation is necessary before transformation. Downstream 

manipulation (e.g, mutagenesis, subsequent insert addition) is facilitated by the minimal vector size 

(1.7-2.0 kb). 

Stabilized Inserts and Gap-Free Cloning 

All CloneSmart cloning kits include the transcription- and translation-free pSMART® vectors, which 

eliminate many of the problems associated with cloning recalcitrant DNA. Conventional cloning 

vectors contain promoters (e.g., lacZ promoter) that constitutively transcribe the insert sequence. 

This transcription and coupled translation can destabilize inserts that contain coding regions, strong 

promoters, short repeats, or incompatible secondary structures. The cloning site of pSMART vectors 

does not include a lacZ sequence. In addition, strong transcription terminators flank the cloning site to 

block spurious transcription from the vector and insert-driven transcription into the vector. Low copy 

number versions of pSMART further stabilize sequences that are difficult to maintain in typical vectors. 

No Background Problems 

The pSMART vectors are supplied pre-digested, with blunt, dephosphorylated ends, and are qualified 

to produce 99.5% recombinant clones in typical experiments. The ultra-low background of empty vec- 

tor (less than 0.5%) eliminates the need to screen for recombinants. It removes the uncertainty of false 

negatives (light blue pUC colonies) and false positives (white colonies that lack inserts), and it enables 

library construction from nanogram amounts of DNA. 

Convenient Success 

Efficiently obtain your clone or create your DNA library—the CloneSmart Cloning Kits eliminate 

tedious preparation of vectors and competent cells, as well as time-consuming QC testing. Kits include 

optimized reagents, ligation-ready vector (no post-ligation cleanup step required), highly efficient 

electrocompetent cells (up to >4 × 10 10 cfu/µg) or chemically competent cells (>1 × 10 9 cfu/µg), detailed 

instructions, and trouble-shooting guides to simplify cloning and sequencing. 


Kan 

ROP Amp 

ROP Kan 

Amp 

Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

Ori 

pSMART ® LCAmp 

2025 bp 

Ori 

pSMART ® 

HCAmp 

1833 bp 

Ori 

Ori 

pSMART ® LCKan 

1980 bp 

pSMART ® 

HCKan 

1788 bp 

SL1 

primer 

SR2 

primer 

SL1 

primer 

SR2 

primer 

SL1 

primer 

SR2 

primer 

SL1 

primer 

SR2 

primer 

EcoRV 

EcoRI 

Blunt 

Blunt 

EcoRI 

XbaI 

EcoRV 

SwaI 

EcoRV 

EcoRI 

Blunt 

Blunt 

EcoRI 

XbaI 

EcoRV 

SwaI 

EcoRV 

EcoRI 

Blunt 

Blunt 

EcoRI 

XbaI 

EcoRV 

SwaI 

EcoRV 

EcoRI 

Blunt 

Blunt 

EcoRI 

XbaI 

EcoRV 

SwaI 




27 


2


ORDERING INFORMATION 

CloneSmart Blunt Cloning Kits contain: Vector 

Premix, CloneSmart DNA Ligase, Positive Control 

Insert DNA, Sequencing Primers, and a complete 

protocol. Kits with cells also include a choice of: 

• E. cloni® ELITE 10G or 10GF' (≥ 2 × 10 10 cfu/µg) 

• 10G SUPREME (≥ 4 × 10 10 cfu/µg) 

Electrocompetent Cells 

• 10G Chemically Competent Cells 

(≥ 1 × 10 9 cfu/µg) 

• 10GF' (≥ 5 × 10 8 cfu/µg) 

Packaged in one (SOLO) or two (DUO) 

transformations per tube. Recovery Medium and 

Control pUC DNA. Kits without cells can be used 

with any E. coli chemically competent or 

electrocompetent cells. 

SIZE DEFINITIONS 

2 

SOLOs: 1 transformation per tube 

DUOs: 2 transformations per tube 

28 Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 


Products 

Kanamycin-Resistant Vector Kits 

CloneSmart HCKan 

Blunt Cloning Kit 

CloneSmart LCKan 


Ampicillin-Resistant Vector Kits 

CloneSmart HCAmp 


CloneSmart LCAmp 





Cells Provided w/Kit Size Cat. No. 

with 10G ELITE 

Electrocompetent cells (SOLOs) 


Electrocompetent cells (DUOs) 

with 10G SUPREME 




with 10G Chemically Competent cells 

(SOLOs) 


(DUOs) 


10 reactions 

20 reactions 

10 reactions 

20 reactions 

10 reactions 

20 reactions 

10 reactions 

20 reactions 

10 reactions 

20 reactions 

10 reactions 

20 reactions 

without cells 20 reactions 

40 reactions 










(SOLOs) 


(DUOs) 

10 reactions 

20 reactions 

10 reactions 

20 reactions 

10 reactions 

20 reactions 

10 reactions 

20 reactions 

10 reactions 

20 reactions 

10 reactions 

20 reactions 


40 reactions 










(SOLOs) 


(DUOs) 

10 reactions 

20 reactions 

10 reactions 

20 reactions 

10 reactions 

20 reactions 

10 reactions 

20 reactions 

10 reactions 

20 reactions 

10 reactions 

20 reactions 


40 reactions 










(SOLOs) 


(DUOs) 

10 reactions 

20 reactions 

10 reactions 

20 reactions 

10 reactions 

20 reactions 

10 reactions 

20 reactions 

10 reactions 

20 reactions 

10 reactions 

20 reactions 


40 reactions 

40708-1 

40708-2 

40706-1 

40706-2 

40719-1 

40719-2 

40717-1 

40717-2 

40730-1 

40730-2 

40728-1 

40728-2 

40704-2 

40704-4 

40834-1 

40834-2 

40832-1 

40832-2 

40845-1 

40845-2 

40843-1 

40843-2 

40856-1 

40856-2 

40854-1 

40854-2 

40821-2 

40821-4 

40054-1 

40054-2 

40052-1 

40052-2 

40065-1 

40065-2 

40063-1 

40063-2 

40076-1 

40076-2 

40074-1 

40074-2 

40041-2 

40041-4 

40313-1 

40313-2 

40311-1 

40311-2 

40324-1 

40324-2 

40322-1 

40322-2 

40335-1 

40335-2 

40333-1 

40333-2 

40300-2 

40300-4


pEZ Seq Cloning Kits 

• Convenient, pre-processed vectors eliminate the need 

for digestion, gel purification, and dephosphorylation. 

• Transcription terminators flanking the cloning site stabilize 

otherwise toxic inserts. 

• Choice of antibiotic resistance. 

• Available with a choice of competent cell transformation 

efficiencies. 

Clone quickly and easy manipulation. 

Lucigen’s unique cloning kits with pre-processed vectors and competent cells eliminate hours of tedious 

preparation and enable highly efficient cloning. Ligation can be completed in as little as 30 minutes 

with no clean-up needed. Only a brief heat denaturation is necessary before transformation. 

The pEZSeq vector (Figure 1) is optimized for high efficiency blunt-end cloning in a blue/white screen- 

ing vector. It is the only commercial preparation of a blue/white screening vector that gives 99% recom- 

binant colonies; common pUC based vectors often produce as many as 30-60% non-recombinant blue 

colonies. The low background of blue colonies with the pEZSeq kit virtually eliminates robotic picking 

errors and greatly simplifies manual clone selection. pEZSeq is small (2,056 bp) and contains little non- 

essential sequence, so unwanted transposition events are minimized. This small vector design facilitates 

cloning larger inserts and simplifies subsequent manipulations. For example, large inserts can be rapidly 

sequenced with transposon insertional sequencing. 

Products 

Kanamycin-Resistant Vector Kit 

pEZSeq HCKan 

Blunt Cloning Kit Kit 

Ampicillin-Resistant Vector Kits 

pEZSeq HCAmp 

Blunt Cloning Kit Kit 






with 

with 

10GF´ 

10GF´ 

ELITE 

ELITE 

Electrocompetent 


cells 

cells 

(DUOs) 

(DUOs) 

with 10G Chemically Competent 

cells (DUOs) 

with with 10GF´ Chemically Competent 

cells cells (DUOs) 

10 10 reactions 


10 10 reactions reactions 


10 

10 

reactions 

reactions 

20 

20 

reactions 

reactions 






40501-1 

40501-2 

40512-1 

40512-2 

40524-1 

40524-2 

40514-1 

40514-2 

40526-1 

40526-2 

without cells 20 20 reactions 40500-2 40500-2 

with 10G ELITE Electrocompetent 


cells (DUOs) 



with 10GF´ ELITE Electrocompetent 

cells (DUOs) 

with 10G Chemically Competent 

cells (DUOs) 

with 10GF´ Chemically Competent 

cells (DUOs) 











40475-1 

40475-2 

40486-1 

40486-2 

40494-1 

40494-2 

40488-1 

40488-2 

40496-1 

40496-2 

40501-1 

40501-2 

40512-1 

40512-2 

40524-1 

40524-2 

40514-1 

40514-2 

40526-1 

40526-2 

40475-1 

40475-2 

40486-1 

40486-2 

40494-1 

40494-2 

40488-1 

40488-2 

40496-1 

40496-2 

without cells 20 20 reactions reactions 40464-2 40464-2 

Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

Figure 1. pEZSeq map. Kan - kanamycin resistance 

gene; Amp - ampicillin resistance gene; Ori - origin of 

replication. Available in high copy only. 


pEZSeq Cloning Kits contain: Vector Premix, 

CloneSmart® DNA Ligase, Positive Control Insert 

DNA, Sequencing Primers, and a complete protocol. 

Kits with cells include 

• E. cloni® ELITE 10G or 10GF' 

(≥ 2 × 10 10 cfu/µg) 

• 10G SUPREME (≥ 4 × 10 10 cfu/µg) 


• 10G Chemically Competent Cells 

(≥ 1 × 10 9 cfu/µg) 

• 10GF' (≥ 5 × 10 8 cfu/µg) 

Packaged in one (SOLO) or two (DUO) 

transformations per tube. Also with Recovery 

Medium and Control pUC DNA. Kits without cells 

can be used with any E. coli chemically competent or 





29 


2


2 

Vector 

Figure 2. GC Cloning. EconoTaq or other non-proofreading 

DNA polymerase adds a single G overhang to the 

PCR products. Ligation to the complementary C-overhang 

pSMART GC Vector is fast and highly efficient. 

Ori 

Figure 3. Schematic diagrams of the GC Cloning vectors. 

Ori - origin of replication; Kan - Kanamycin resistance 

gene; ROP - Repressor of priming. 

Positions of transcription terminators (T) are indicated. 

CFU/Ligation 

G 

C 

2.00E+06 

1.60E+06 

1.20E+06 

8.00E+05 

4.00E+05 

0.00E+00 

G 

5´ 

3´ 

INSERT G 

3´ 

5´ 

C 

SL1 primer SR2 primer 

C 

pSMART ® GC HK 

Terminator 

GC Cloning vs TOPO TA Cloning 

Total CFU 

30 min Ligation 

GC HK GCBlue TopoTA 

Figure 4. GC Cloning vs. TOPO TA cloning. Each vector 

was ligated to a chloramphenicol-resistant expression 

cassette directly from a PCR reaction using manufacturers’ 

protocols. White colonies from kanamycin plates 

were patched onto chloramphenicol plates to determine 

the fraction with correct CmR inserts. 

30 Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

Kan 

PCR 

Product 

C 

5´ 

3´ 

3´ 

5´ 

G 

INSERT 

C 

G 

G 

SL1 primer SR2 primer 

Ori ROP 

C 

pSMART ® GC LK 

Kan 

White CmR CFU 

White Cm-Sensitive CFU 

Blue CFU 

5 min Ligation 

Vector 

Ori 

PCR CLONING 

GC Cloning and Amplification (pSMART® GC Vectors) 

• Clone PCR products from proofreading or non-proofreading 

polymerases. 

• Transcription terminators flanking the cloning site stabilize otherwise 

unclonable inserts. 

• No lost clones or deleted sequences. 

Advanced PCR cloning 

GC Cloning (U.S. Patent 7,723,103) is similar to TA-cloning (Mead, D., et. al. 1991.) TA-cloning takes 

advantage of the well-known property of non-proofreading DNA polymerases (e.g., Taq, Tfl, Tth) to 

add a single 3′-A to PCR products. 

The GC Cloning technology is based on the discovery that these same enzymes add a single 3′-G to 

INSERT G 

G 

C DNA molecules, either during PCR or as a separate G-tailing reaction to any blunt DNA (Figure 2). The 

lacZ 

pSMART® GC vectors (Figure 3) contain a single 3′-C overhang, which is compatible with the single 3′-G 

overhang on the inserts. 

pGC Blue 

Superior Performance 

Compared to TOPO TA Cloning, pSMART GC Vectors produce more colonies per ligation and more 

colonies with the correct insert (Figure 4). Furthermore, PCR products in pSMART GC show significantly 

higher stability than the same insert in a TOPO TA vector (Figure 5). 


Efficiently clone your PCR product regardless of its size, base composition, or the enzyme used to 

generate it. Kits include reagents for amplification (GC Cloning and Amplification Kits, only) and ligation, 

ligation-ready vector (no post-ligation cleanup step required), highly efficient electrocompetent cells 

(≥ 4 × 10 10 cfu/µg) or chemically competent cells (≥1 ×10 9 cfu/µg), detailed instructions, and trouble- 

shooting guides. 




C 

plac 

Kan 

MW GC Cloning | TOPO TA MW 

Figure 5. A Thermus species polA gene was amplified and cloned into either the pSMARTGC 

or pcrII TOPO TA vector. All GC clones contained the expected product; deletions and empty 

clones were common in the TOPO TA vector. 


PCR CLONING 

Products 

GC Cloning & Amplification 

Kit (pSMARTGC HK) 


Kit (pSMARTGC LK) 


Kit (pGC Blue) 



with 10G ELITE Electrocompetent Cells 

(SOLOs) 

with 10G SUPREME Electrocompetent 

Cells (SOLOs) 

with 10G Chemically Competent Cells 

(SOLOs) 


(SOLOs) 


Cells (SOLOs) 


(SOLOs) 


(SOLOs) 


Cells (SOLOs) 


(SOLOs) 

GC Cloning & Amplification Kits contain: 4X GC Vector Premix (includes buffer, ATP; and either pSMART ® GC 

HK, pSMARTGC LK, or pGC Blue); CloneSmart ® DNA Ligase; T4 Polynucleotide Kinase; 10X T4 Polynucleotide 

Kinase buffer (containing ATP); EconoTaq DNA Polymerase; EconoTaq 10X Reaction Buffer (with Mg ++ ); Control 

lacZ template plus PCR primers; CloneSmart Sequencing Primers; a choice of E. cloni 10G SUPREME (≥4 x 10 10 cfu/µg) 

or ELITE (≥2 x 10 10 cfu/µg) Electrocompetent Cells, or 10G Chemically Competent Cells (≥1 x 10 8 cfu/µg), in SOLO 

packaging (one transformation per tube); Recovery Medium, and Control pUC DNA. 


10 reactions 

20 reactions 

10 reactions 

20 reactions 

5 reactions 

10 reactions 

20 reactions 

10 reactions 

20 reactions 

10 reactions 

20 reactions 

10 reactions 

20 reactions 

10 reactions 

20 reactions 

10 reactions 

20 reactions 

5 reactions 

10 reactions 

20 reactions 

40731-1 

40731-2 

40732-1 

40732-2 

40733-0 

40733-1 

40733-2 

40736-1 

40736-2 

40737-1 

40737-2 

40738-1 

40738-2 

40742-1 

40742-2 

40741-1 

40741-2 

40743-0 

40743-1 

40743-2 

Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 




31 


2


223 kb 

9 kb 

6 kb 

telN repA 

telN rep e A 

NotI 

AhdI 

ApaI 

(SmaI) 

32 Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

cB 

cB 

Insert DNA 

C 

G 

Insert DNA 

G 

C 

(SmaI) 

AhdI 

NotI 

Kan r 

pJAZZ-OK 

pJAZZ-OK GC 

Figure 6. The pJAZZ-OK Blunt and GC vectors. The left 

arm is 10 kb and the right arm is 2.2 kb. Top Panel – After 

processing, the vector has blunt dephosphorylated ends; 

blunt insert DNA is ligated to vector arms. Bottom panel 

– After processing, the GC vector has C-tailed, dephosphorylated 

ends; G-tailed insert DNA is ligated to vector 

arms. telN - protelomerase gene; repA - replication 

factor and origin of replication; cB - regulator of copy 

number; Kanr - kanamycin resistance gene. Approximate 

position of transcription terminators (T) are indicated. 

Figure 7. Large PCR products cloned into BigEasy Long 

PCR Cloning Kit. The Left Arm (10 kb) is indicated; the 

Right arm was run off the gel. 

Kan r 

10 kb 

8 kb 

6 kb 

PCR CLONING 

BigEasy® Long PCR Cloning Kit 

• Clone any long or short PCR product. 

• Clone PCR products from proofreading or non-proofreading 

polymerases. 

• Stabilize otherwise unclonable inserts. 

Advanced PCR cloning 

GC Cloning (U.S. Patent 7,723,103) is the first major advance in PCR cloning since the first description 

of TA-cloning (Mead, et. al. 1991.) TA-cloning takes advantage of the well-known property of non- 

proofreading DNA polymerases (e.g., Taq, Tfl, Tth) to add a single 3´-A to PCR products. 

The GC Cloning technology is based on the discovery that these same enzymes add a single 3´-G to 

DNA molecules, either during PCR or as a separate G-tailing reaction to any blunt DNA (Figure 2). The 

BigEasy Long PCR Cloning Kits contain everything needed to efficiently clone blunt or G-tailed PCR 

products up to 30 kb into the unbiased, high-fidelity pJAZZ linear cloning vector. There are two ver- 

sions of the vector (Figure 6) that are compatible with either proofreading or non-proofreading PCR 

polymerases. 

Stabilized inserts 

The pJAZZ vector is ideal for cloning difficult PCR products of any size up to 30 kb (Figure 7). Because 

the pJAZZ vector is linear, the ends of the vector can rotate freely. As a result, the vector does not 

supercoil. Without the torsional stress induced by supercoiling, otherwise unclonable sequences (e.g., 

repetitive, or A/T- or G/C-rich sequences) are stabilized. The pJAZZ linear cloning vector is main- 

tained at low copy number (5-10/cell) in BigEasy-TSA Electrocompetent Cells, which are required for 

transformation and propagation. The copy number can be induced 5-10X in this strain. In addition, the 

pJAZZ vector incorporates Lucigen’s CloneSmart® technology for transcription-free cloning, which fur- 

ther increases insert stability. The pJAZZ vector can be isolated using standard plasmid prep methods. 


Efficiently clone your PCR product regardless of its size, base composition, or the enzyme used to gen- 

erate it. Kits include ligase and buffer (no post-ligation cleanup step required), and highly efficient TSA 

electrocompetent cells (≥4 × 10 10 cfu/µg), detailed instructions, and trouble-shooting guides. 

Products 

BigEasy Long PCR Cloning Kit (pJAZZ-OK Blunt) 5 reactions 

10 reactions 

20 reactions 

BigEasy Long PCR Cloning Kit (pJAZZ-OK GC) 5 reactions 

10 reactions 

20 reactions 

BigEasy-TSA Electrocompetent Cells 

(> 4 × 10 10 cfu/mg) (SOLOs) 

Order Information 

The BigEasy ® Long PCR Cloning Kit contains: pre-cut vector pJAZZ ® -OK (Blunt or C-tailed overhang), CloneSmart ® 

DNA Ligase, CloneDirect 10X Ligation Buffer, PCR Control Template and Primers, Sequencing Primers, T4 

Polynucleotide Kinase, 10X Primer Kinase Buffer, BigEasy-TSA Electrocompetent Cells, Positive Control Plasmid, 

Recovery Medium, Arabinose Induction Solution, YT Agar Powder, and complete protocols. 





Size Cat. No. 

6 reactions 

12 reactions 

24 reactions 

43054-1 

43054-2 

43054-3 

43066-1 

43066-2 

43066-3 

60224-1 

60224-2 

60224-3

BAC, FOSMID, LARGE, OR DIFFICULT CLONING 

CopyRight® v2.0 BAC Cloning Kits 

(pSMART® BAC and pEZ BAC) 

• Efficiently clone any size insert up to 200kb. 

• Create libraries from A/T- or G/C-rich genomes. 

• Clone gene clusters or operons. 

• High insert stability & Inducible copy number. 

Lucigen's CopyRight® v2.0 BAC cloning kits offer unprecedented accuracy, reliability, and efficiency 

in BAC cloning and library construction. The pSMART® BAC or pEZ BAC vectors incorporate the 

CloneSmart® transcription-free technology to minimize cloning bias and enable cloning of otherwise 

toxic genes (Figure 8) and BAC-Optimized Replicator v2.0 Electrocompetent Cells along with induc- 

ible copy number amplification (20-50X) for high DNA yields and a lacZ-sacB stuffer fragment that 

eliminates uncut vector from recombinants, resulting in zero background. Unlike other BAC cloning 

systems, Lucigen's CloneSmart technology assures that even "unclonable" inserts are stably maintained. 

Sequences are not lost or rearranged in the cloning process. CopyRight Kits offer superior perfor- 

mance for BAC cloning at a much better price than other kits. Figure 9 shows an example of a Random 

Shear BAC library generated with pSMART BAC. 


Efficiently obtain your clone or create your library— 

the CopyRight v2.0 Cloning Kits eliminate tedious 

vector and competent cell preparation, as well as 

time-consuming QC experiments. Kits include opti- 

mized reagents, ligation-ready vector, highly efficient 

electrocompetent cells, detailed instructions, and a 

trouble-shooting guide. 

Products 

CopyRight v2.0 pEZ BAC 


CopyRight v2.0 pEZ BAC 

BamHI Cloning Kit 

CopyRight v2.0 BAC 

Cloning Kit, pSMART 

BAC BamHI 



BAC EcoRI 



BAC HindIII 

CopyRight v2.0 Fosmid 

Cloning Kit 

Figure 9. Arabidopsis Random Shear BAC Library in 

the pSMART BAC vector. 7,680 clones were obtained, 

with an average insert size of 105 kb and ~5X genome 

coverage. 


with Replicator v2.0 Electrocompetent 

Cells (DUOs) 

10 reactions 

20 reactions 


42009-1 

42009-2 

without cells 10 reactions 42016-1 


Cells (DUOs) 

10 reactions 

20 reactions 

42007-1 

42007-2 

without cells 10 reactions 42012-1 


Cells (DUOs) 


Cells (DUOs) 


Cells (DUOs) 

BAC-Optimized Replicator v2.0 Electrocompetent Cells 

(≥ 2 × 10 10 cfu/µg) 

10 reactions 

20 reactions 

10 reactions 

20 reactions 

10 reactions 

20 reactions 

5 reactions 

10 reactions 

20 reactions 

12 reactions 

24 reactions 

42030-1 

42030-2 

42031-1 

42031-2 

42032-1 

42032-2 

42027-1 

42027-2 

42027-3 

60210-1 

60210-2 

Inc C 

Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

Inc C 

cosN 

ori 2 

ori V 

pSMART ® BAC 

7.5 kb 

par A rep E 

par B par C 

Terminator 

Figure 8. CopyRight vectors pSMART BAC and pEZ 

BAC. ori2, repE, IncC - single-copy origin of replication; 

oriV - inducible origin of replication; par A,B,C 

- partition genes; Cm r - chloramphenicol-resistance 

gene; cosN - lambda packaging signal; T - CloneSmart 

transcription terminators; sacB -sucrase gene lacZ 

- alpha peptide of the beta galactosidase gene. Approximate 

positions of sequencing primers and transcription 

terminators are indicated. 


rep E par A par B par C 

SL1 primer 

SR4 primer 

Cmr 

pEZ BAC 

7.2 kb 

ori 

Terminator 

Terminator 

ori V lacZ 

Cm 

Not I 

Ahd I 

Pml I 

EcoR I 

Hind III 

BamH I 

BstX I 

Stuffer 3.3Kb 

BstX I 

BamH I 

Hind III 

EcoR I 

Pml I 

Ahd I 

Not I 

Each CopyRight® v2.0 Cloning Kit contains: pre-cut 

CopyRight vector (pEZ BAC BamHI or pEZ BAC Blunt 

or pSMART® BAC BamHI, EcoRI, or HindIII), CopyRight 

DNA Ligase, Copyright 5X Ligation Buffer (includes 

ATP), Positive Control Insert DNA, Arabinose Induction 

Solution, Sequencing Primers, and complete protocols. Kits 

with cells also include BAC-Optimized Replicator v2.0 

Electrocompetent Cells (≥2 × 10 10 cfu/µg DNA) packaged 

in two transformations per tube (DUOs), Positive Control 

Plasmid, YT Agar Powder, and Recovery Medium. BAC- 

Optimized Replicator v2.0 Electrocompetent Cells are also 

available separately. 

BAC-Optimized Replicator v2.0 Electrocompetent Cells 

contain trfA gene necessary for conditional amplification of 

vector copy number. If copy amplification is not needed, 

CopyRight Kits can be used with Lucigen’s E. cloni® 10G BAC- 

Optimized Electrocompetent Cells (p. 44), or with any E. coli 

competent cells. 

Each CopyRight v2.0 Fosmid Cloning Kit contains: pre-cut, 

dephosphorylated pSMART FOS blunt vector, CloneSmart 

DNA Ligase, CloneDirect 10X Ligation Buffer (includes 

ATP), DNATerminator End Repair Enzyme Mix and End 

Repair Buffer, Replicator FOS strain (glycerol stock), 20% 

Maltose Solution, 1M MgSO 4 , SM Buffer, Arabinose Induction 

Solution, Sequencing Primers, and complete protocols. The 

20-reaction Kit is provided as two 10-reaction Kits. 

sacB lacZ 

Terminator 

AscI 

NotI 

lox BEZ-F primer 

HindIII 

SphI 

HpaI 

BamHI 

PmlI 

T7 EcoRI 

BEZ-F primer 

NotI 

PmeI 

cosN 

r lacZ 




33 


2


2 

tel N repA 

tel N repA 

Figure 10. pJAZZ-OC and pJAZZ-OK linear vectors. 

RepA, replication factor and low copy origin of replication 

(~2-4 per cell; inducible 5-10 fold); Camr - chloramphenicol 

resistance gene; Kanr - kanamycin resistant 

gene; telN - protelomerase gene; cB - replication regulator. 

Approximate positions of transcription terminators 

(T) are indicated. 

96% AT rich DNA 

NotI 

AhdI 

ApaI 

(SmaI) 

34 Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

cB 

cB 

C 

G 

6-20 kb Oxytricha inserts 

10 kb 

8 

6 

3 

2 

1 

Insert DNA 

G 

Insert DNA 

C 

(SmaI) 

AhdI 

NotI 

Cam r 

Figure 12. Oxytricha trifallax genomic DNA (75-85% AT) 

was sheared to 6-20 kb and cloned into the pJAZZ linear 

vector. NotI digests of mini-prep DNA are shown. 

Kan r 

pJAZZ-OK 

pJAZZ-OK GC 

BAC, FOSMID, LARGE, OR DIFFICULT CLONING 

BigEasy® v2.0 Linear Cloning System 

• Maximum insert stability. 

• Efficiently clone any insert up to 30 kb. 

• Create libraries from A/T-rich or G/C-rich genomes. 

• Clone gene clusters or operons. 

• Inducible copy number. 

The BigEasy Kit with the pJAZZ vector is ideal for constructing bias-free, large-insert genomic libraries, 

or for cloning difficult DNA of any size up to 30 kb. Because the novel pJAZZ vector is maintained as 

a linear molecule, the ends of the vector can rotate freely (Fig. 10). As a result, the vector does not 

supercoil. Without the torsional stress induced by supercoiling, otherwise unclonable sequences 

(e.g., repetitive, or A/T-rich or G/C-rich sequences) are stabilized (Fig. 11). The pJAZZ linear cloning 

vector is maintained at low copy number (5-10/cell) in BigEasy-TSA Electrocompetent Cells, which are 

required for transformation and propagation. The copy number can be induced 5-10X in this strain. In 

addition, the pJAZZ vector incorporates Lucigen’s CloneSmart® technology for transcription-free clon- 

ing, which further increases insert stability. The pJAZZ vector can be isolated using standard plasmid 

prep methods. 


The BigEasy v2.0 Kits eliminate tedious vector and competent cell preparation, as well as time-consum- 

ing QC testing. Kits include optimized reagents, ligation-ready vector, highly efficient electrocompe- 

tent cells, detailed instructions, and trouble-shooting guides. 

Products 

BigEasy v2.0 Linear Cloning Kit (pJAZZ-OC Blunt Vector) 

w/BigEasy-TSA Electrocompetent Cells (SOLOs) 

BigEasy v2.0 Linear Cloning Kit (pJAZZ-OC NotI Vector) 


BigEasy-TSA Electrocompetent Cells 

(> 4 x 10 10 cfu/mg) (SOLOs) 

BigEasy v2.0 Linear Cloning Kit (pJAZZ-OK Blunt Vector) 


BigEasy v2.0 Linear Cloning Kit (pJAZZ-OK NotI Vector) 



The BigEasy ® Linear Cloning Kit includes: Dephosphorylated pJAZZ ® Vector pre-cut at either a SmaI (blunt) or NotI 

site, CloneSmart ® DNA Ligase, CloneDirect 10X Ligation Buffer (includes ATP), DNATerminator ® End Repair 

Enzyme & 5X End Repair Buffer (Blunt Kit only), Sequencing Primers, Positive Control Insert DNA, BigEasy-TSA 

Electrocompetent Cells in SOLO packaging (1 transformation per tube), Recovery Medium, Transformation 

Control DNA, and complete protocols. BigEasy-TSA Electrocompetent Cells are also available separately. 





Size Cat. No. 

5 reactions 

10 reactions 

20 reactions 

5 reactions 

10 reactions 

20 reactions 

6 reactions 

12 reactions 

24 reactions 

5 reactions 

10 reactions 

20 reactions 

5 reactions 

10 reactions 

20 reactions 

43018-1 

43018-2 

43018-3 

43024-1 

43024-2 

43024-3 

60224-1 

60224-2 

60224-3 

43036-1 

43036-2 

43036-3 

43042-1 

43042-2 

43042-3

ACCESSORY KITS FOR CLONING 

DNATerminator® End Repair Kit 

• Repair sheared or restriction-digested DNA ends 

• Increase cloning efficiency as much as 5-fold 

• Optimized for consistent & reliable results 

Repair DNA ends 

Double-stranded DNA fragments for library construction or large-scale 

sequencing are often generated by mechanically shearing large DNA (e.g. 

genomic or BAC), a process that primarily leaves uneven ends. Large DNA 

may also be cut with restriction enzymes to generate smaller fragments for 

subcloning. Sheared or restricted DNA fragments must be “end-repaired” 

before ligation into blunt-end cloning vectors (e.g., Lucigen’s CloneSmart®, 

CopyRight®, or BigEasy® kits). 

The DNATerminator End Repair Kit creates blunt, 5' phosphorylated ends 

on any type of sheared or restriction-digested DNA fragment, ensuring the 

highest possible cloning efficiency. 

Products 


Size Cat. No. 

DNATerminator ® End Repair Kit 10 reactions 40035-1 



The DNATerminator End Repair Kit contains: End repair Enzyme Mix, End Repair 

Buffer, and a complete protocol. 


PCRTerminator® End Repair Kit 

• Optimizes cloning of any PCR product into blunt vectors 

• Obtain higher cloning efficiency 

• Easy to use 

Polish the ends of PCR products 

PCR products generated with non-proofreading polymerases such as Taq, 

Tth, or Tfl primarily have single 3´ G- or 3´ A-overhangs. These products 

can be treated with the PCRTerminator Kit for cloning into blunt vectors 

(e.g., Lucigen’s CloneSmart®, CopyRight®, or BigEasy® systems). The 

PCRTerminator Kit also adds 5´ phosphates to amplification products, which 

is essential for cloning PCR products from non-proofreading or proofread- 

ing polymerases (such as Pfu or Vent®) into dephosphorylated vectors. 

Products 


The PCRTerminator End Repair Kit contains: End Repair Enzyme Mix, End Repair 

Buffer, and a complete protocol. 

Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

Size Cat. No. 

PCRTerminator ® End Repair Kit 10 reactions 40037-1 






35 


2


2 

36 Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 


UltraClone DNA Ligation and Transformation Kits 

• Reagents for ligation and transformation. 

• Fast, simple procedure. 

• 10X more recombinants from every reaction! 

Maximize recombinant clones 

Products 


UltraClone DNA Ligation & Transformation Kit contains: 10X CloneDirect Buffer (includes ATP), CloneSmart 

engineered DNA Ligase, and a choice of: E. cloni ELITE 10G or 10GF' (≥ 2 × 10 10 cfu/µg) or 10G SUPREME 

(≥ 4 × 10 10 cfu/µg) Electrocompetent Cells, or 10G Chemically Competent Cells (≥ 1 × 10 8 cfu/µg), in DUO 

(2 transformations per tube) or SixPack (6 transformations per tube). Packaging, as indicated, Recovery Medium, 

and Transformation Control DNA. 

CloneDirect Rapid Ligation Kit 

• Ligation reaction is transformation-ready in minutes 

• No DNA cleanup step required 

• Highly reliable and reproducible results 

Products 


The CloneDirect Rapid Ligation Kit contains: 10X CloneDirect Buffer (contains ATP), CloneSmart ® DNA Ligase, and 

complete protocols. 

See web site for additional products. 





UltraClone Kit with 10G SUPREME Electrocompetent 

Cells (DUOs) 


(DUOs) 


(SixPacks) 

with 10GF´ ELITE Electrocompetent Cells 

(DUOs) 


(DUOs) 












Size Cat. No. 

CloneDirect Rapid Ligation Kit 24 reactions 40020-2 


96 reactions 40020-5


Gel-Ready DNA Ladders 

• Bench-top stable markers for gel electrophoresis. 

• Choice of low or high molecular weight range. 

DNA ladders: ready when you are 

Gel-Ready DNA Ladders are precise, ready-to-use double-stranded DNA markers for on-gel size and 

mass determination. Available as 1-kb and 100-bp DNA Ladders, these markers cover a convenient 

size range. Discrete bands of higher intensity provide easy size reference. 

Products 

Size Cat. No. 

Gel-Ready 100bp DNA Ladder 100 gel lanes 50020-1 

Gel-Ready 1kb DNA Ladder 100 gel lanes 50010-1 


Each DNA ladder is supplied as 1 ml of premixed DNA ladder (0.5 µg/10 µl) in loading buffer (10 mM EDTA, 

10% glycerol, 0.015% bromophenol blue, and 0.17% SDS). 

Bst Adapter Fill-In Kit 

• Close gaps remaining after linker or adapter ligation. 

• Repair nicked DNA. 

Next Generation (NexGen) Sequencing, PCR amplification of damaged DNA, and other applications in 

molecular biology result in nicks or gaps in DNA. Ligation of non-phosphorylated adapters (or linkers) 

in NexGen sequencing leaves a single-strand nick that would prevent subsequent amplification of the 

internal sequence. The Bst Adapter Fill-In Kit combines the strand displacement polymerase from Bacillus 

stearothermophilus (Bst) with an optimized buffer containing nucleotides to eliminate nicks and gaps 

in sample DNA. 

Products 

Size Cat. No. 

Bst Adapter Fill-In Kit 10 rxns 40031-1 


The Bst Adapter Fill-In Kit combines the strand displacement polymerase from Bacillus stearothermophilus (Bst) 

with an optimized buffer containing nucleotides. 


Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

1 kb 

DNA Ladder 

bp 

10000 

8000 

6000 

5000 

4000 

3000 

2500 

2000 

1500 

1000 

700 

500 

300 

Gel-Ready DNA Ladders 

100 bp 

DNA Ladder 

bp 

1000 

900 

800 

700 

600 

500 

400 

300 

200 

100 

Figure 13. Size of each band in the Gel- 

Ready 1-kb and 100-bp DNA Ladders. 

Higher intensity bands are at 5 kb and 

500 bp. 

50 




37 


2

SECTION 3 


Overview ..........................................40 

General Cloning & 

Library Construction .......................42 

Custom Competent Cells ...............44 

Phage Display Library Apps ...........45 

Large/Difficult Fragment Cloning ..46 


Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

Solutions 




39


3 

OVERVIEW 


General Cloning and Library Construction 

E. cloni® 10G & 10GF’ Cells » Construct libraries, perform general cloning, pro- 

duces ssDNA. Clone, subclone, propagate plasmids. Transform using heat shock. 

E. cloni® 5-alpha Chemically Competent Cells » Clone, subclone. Trans- 

form using heat shock. 

Large or Difficult Insert Cloning 

Endura Competent Cells, BAC-Optimized Replicator v2.0 & 10G BAC-Opti- 

mized Electrocomp, BigEasy® TSA Electrocompetent Cells 

Table 1. Lucigen Competent Cells for cloning 

Phage Display Cells 

Cell Lines 

40 Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 




Phage Display Library Applications 

SS320 Electrocompetent Cells » Produce phage display libraries from cells with 

the highest available transformation efficiency. Not an amber suppressing strain. 

TG1 Electrocompetent Cells » Make phage display libraries and express 

protein. 

Transformation 

Efficiency 

(cfu/µg pUC DNA) 

ER2738 Electrocompetent Cells » Use for phage display libraries including 

Ph.D. Phage Display Libraries. 

Custom Competent Cells 

Any Lucigen strain is available in bulk quantities and custom dispensing. Any E. 

coli strain can be produced as highly efficient electrocompetent cells. 

Cloning 

Methylated DNA 


BAC, Cosmid 

Cloning 

Blue/White 

Screening 

10G SUPREME Electrocompetent >4 × 10 10 ✓ ✓ ✓ 

10G ELITE Electrocompetent >2 × 10 10 ✓ � ✓ 

10G BAC-Optimized Electrocompetent >1 × 10 10 ✓ ✓ ✓ 

10G CLASSIC Electrocompetent >5 × 10 9 ✓ � ✓ 

10G Chemically Competent >1 × 10 9 ✓ � ✓ 

10GF´ ELITE Electrocompetent >2 × 10 10 ✓ � ✓ 

10GF´ Chemically Competent >5 × 10 8 ✓ � ✓ 

BigEasy ® -TSA Electrocompetent >4 × 10 10 ✓ � 

TG1 Electrocompetent >4 × 10 10 ✓ � 

SS320 >4 × 10 10 ✓ � 

ER2738 >2 × 10 10 ✓ � 

5-alpha Chemically Competent >1 × 10 8 � � 

BAC-Optimized Replicator v2.0 Electrocompetent >2 × 10 10 ✓ ✓ 

NEW Endura Chemically Competent Cells 

NEW Endura ElectroCompetent Cells 

See also 

✓ Protein Expression Section p. 55 

Lucigen offers the best performance, value and convenience in competent cells for cloning, genomic and phage display library applications, and protein 

expression. No other supplier offers the spectrum of competent cells for your cloning and expression needs. 

✓ 

without IPTG induction 

✓ 

IPTG induction required 

✓ 


✓ 


✓ 

without IPTG induction 

✓ 


>1 × 10 8 

>1 × 10 10 � � �

Competent Cells Comparison 

LUCIGEN Efficiency * Size LIFE TECHNOLOGIES Efficiency* Size Price/rxn AGILENT (STRATAGENE) Efficiency * Size 

CLONING CELLS - DH5a, DH10B, etc. Substitute 

E. cloni® 10G SUPREME Electrocompetent (DUOs) ≥ 4 × 10 10 

12 rxns 

10 MegaX DH10B Electrocompetent ≥ 3 × 10 25 rxns $11.16 ElectroTen-Blue ® Electrocompetent ≥ 3 × 10 10 10 rxns 

24 rxns 

E. cloni 10G ELITE Electrocompetent (Six Packs) ≥ 2 × 10 10 

24 rxns 

ElectroMAX DHI0B ≥ 1 × 10 


10 25 rxns $9.84 XL-1 Blue Electrocompetent ≥ 1 × 10 10 10 rxns 

48 rxns 

E. cloni 10G CLASSIC Electrocompetent (Six Packs) ≥ 5 × 109 24 rxns 20 rxns $10.40 

9 

TOP10 Electrocompetent ≥ 1 × 10 40 rxns $9.65 

No equivalent. 

48 rxns 

120 rxns $9.22 

E. cloni 10G Chemically Competent (DUOs) ≥ 1 × 109 12 rxns ® One Shot TOP10 

≥ 1 × 10 

Chemically Competent 

9 

10 rxns $21.70 

20 rxns $17.60 

XL-10 Gold 

40 rxns $17.10 

® Ultracompetent ≥ 5 × 109 10 rxns 

24 rxns 

48 rxns 

XL-1 Blue Supercompetent ≥ 1 × 109 Max Efficiency 

10 rxns 

® DH10B ChemComp ≥ 1 × 109 96 rxns 

20 rxns $11.70 

E. cloni 10G Chemically Competent 

≥ 1 × 10 

(Subcloning Grade) 

6 

48 rxns Subcloning Efficiency DH5a 

≥ 1 × 10 


6 XL1-Blue Subcloning-Grade 

40 rxns $1.88 

≥ 1 × 10 


6 80 rxns 

96 rxns 

GENERAL CLONING AND LIBRARY CONSTRUCTION 

≥ 1 × 10 9 20 rxns $9.35 No equivalent. 

E. cloni 5-alpha Chemically Competent Cells (DUOs) ≥1 × 10 8 12 rxns Max Efficiency ® DH5a Chemically 

Competent 

24 rxns 

DIFFICULT/LENTIVIRAL CLONING 

10 rxns 

10 rxns 

≥ 5 × 108 ≥ 1 × 109 SURE Chemically Competent 

SURE2 Supercompetent 

12 rxns OneShot Stbl3 ≥ 1 × 10 8 20 rxns $17.80 

Endura Chemically Competent (DUOs) ≥ 1 × 10 8 

24 rxns 


12 rxns 

ElectroMax Stbl4 ≥ 5 × 10 9 25 rxns $10.08 SURE Electrocompetent ≥ 1 × 10 10 10 rxns 

Endura Electrocompetent (DUOs) ≥ 1 × 10 10 

24 rxns 

PHAGE DISPLAY 

TG1 Electrocompetent Cells (DUOs) ≥ 4 × 10 10 12 rxns 

No equivalent. TG1 Electrocompetent Cells ≥ 1 × 10 10 10 rxns 

24 rxns 

SS320 (MC1061 F′) Electrocompetent Cells (DUOs) ≥ 4 × 10 10 

12 rxns 

No equivalent. No equivalent. 

24 rxns 

ER2738 Electrocompetent Cells (DUOs) ≥ 2 × 10 10 

12 rxns 


24 rxns 

PROTEIN EXPRESSION BL21(DE3) CELLS - Also available in pLysS and pLysE versions, as well as a ComboPack containing 4 transformations of all 3 strains 

E. cloni EXPRESS BL21(DE3) Electrocompetent ≥ 5 × 109 12 rxns 


24 rxns 

E. cloni EXPRESS BL21(DE3) Chemically Competent ≥ 1 × 107 12 rxns 

One Shot BL21(DE3) Chemcomp 1 × 108 BL21-Gold 

20 rxns $12.45 

® (DE3) Chemcomp ≥ 1 × 108 10 rxns 

24 rxns 

BL21(DE3) Chemcomp ≥ 1 × 106 48 rxns 

10 rxns 

Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

TOXIC PROTEIN EXPRESSION - OverExpress Cells - Also available in pLysS versions, as well as a ComboPack containing 3 transformations of all 4 strains 



OverExpress C41(DE3) Electrocompetent ≥ 1 × 10 10 12 rxns 

24 rxns 

OverExpress C43(DE3) Electrocompetent ≥ 1 × 10 10 12 rxns 

24 rxns 

OverExpress C41(DE3) Chemically Competent ≥ 1 × 106 12 rxns 

24 rxns 

OverExpress C43(DE3) Chemically Competent ≥ 1 × 106 12 rxns 

24 rxns 




41 


3


3 

CFU x 10 10 

7.0 

5.0 

3.0 

1.0 

0.0 

Recombinant Yields 

Lucigen Competitor’s 

10G SUPREME "ultra high efficiency” cells 

Figure 1. E. cloni 10G SUPREME Electrocompetent Cells consistently 

outperformed “ultra high efficiency” cells from a 

leading supplier. Both strains were transformed with 

10 pg of pUC19 (n=16). 

42 Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

E. cloni® 10G & 10GF’ 

Electrocompetent & Chemically Competent Cells 

Construct libraries, perform general cloning, produce ssDNA. 

• Direct replacements for standard cloning strains 

(e.g., DH5α, DH10B, JM109, TOP10, XL1-Blue, etc.) 

• Optimized genetics for high yields: phage T1 resistant, endonuclease and 

recombination minus, blue/white screening-capable. 

• Available in a range of high transformation efficiencies 

(5 × 10 9 to 4 × 10 10 cfu/μg). 

• Convenient packaging options. 

Cloning Strain Comparison 

• E. cloni 10G SUPREME Electrocompetent Cells (≥ 4 × 10 10 cfu/µg pUC 

DNA) » SUPREME Cells have the highest transformation efficiency avail- 

able from any supplier and provide recombinant yields higher than the 

highest efficiency cells offered by a leading supplier (Figures 1). Choose 

SUPRE ME Cells for the most demanding cloning situations, such as 

construction of large, high complexity libraries or cloning difficult targets, 

which require the greatest number of transformants possible. 

• E. cloni10G & 10GF´ ELITE Electrocompetent Cells (≥ 2 × 10 10 

cfu/µg pUC DNA). » ELITE Cells have twice the transformation efficiency 

compared to “ultra high efficiency” cells from other suppliers. ELITE Cells 

provide large numbers of transformants from hard-to-clone fragments or 

limited DNA at a lower price. 

• E. cloni 10G CLASSIC Electrocompetent Cells (≥ 5 × 10 9 cfu/µg pUC 

DNA). » CLASSIC Cells are high efficiency cells with a substantially lower 

cost per reaction. These cells are the most economical choice for standard 

cloning and library construction. 





Electrocompetent Chemically Competent 

Strain Type Efficiency Type Efficiency 

E. cloni 10G SUPREME > 4 × 10 10 cfu/µg > 1 × 10 9 cfu/µg 

ELITE > 2 × 10 10 cfu/µg 96-well > 1 × 10 8 cfu/µg 

CLASSIC > 5 × 10 9 cfu/µg 


Subcloning Grade > 1 × 10 6 cfu/µg 

E. cloni 10GF' ELITE > 2 × 10 10 cfu/µg > 5 × 10 8 cfu/µg 

Lucigen’s E. cloni competent cells share the most useful genetic elements of standard cloning strains like DH5α, DH10B, JM109, TOP10, etc. and 

directly replace them in cloning protocols. However, E. cloni electrocompetent cells incorporate a unique manufacturing technology that increases 

transformation efficiency, recombinant yields and reliability (Figure 1), while decreasing costs. These cells provide solutions for a wide range of 

applications at economical prices. 

Choice of strain: 

• E. cloni 10G Competent Cells » Library construction, cloning, subcloning, and plasmid isolation with or without blue/white screening. 

• E. cloni 10GF΄ » Competent Cells. Contain the F' plasmid for infection with M13 to produce ssDNA. 

Choice of efficiency: 

• E. cloni 10G Chemically Competent Cells (≥ 1 × 10 9 cfu/µg pUC DNA; 

≥ 1 × 10 8 in 96-well plates). » Highly efficient competent cells for routine 

cloning applications. 

• E. cloni 10GF´ Chemically Competent Cells 

(≥ 5 × 108 cfu/µg pUC DNA). » An excellent choice for producing 

single-stranded DNA for sequencing, mutagenesis, etc. 

• E. cloni 10G Chemically Competent Cells, Subcloning Grade (≥ 1 × 10 6 

cfu/µg pUC DNA). » The best value available anywhere for simple clon- 

ing and plasmid propagation. 

FREE SAMPLE 

lucigen.com/samples


Products 


E. cloni® 10G SUPREME Cells (>4 × 10 10 cfu/µg) 60081-1 

60081-2 


GENOYTPES 

Cat. No. Size 

60080-1 

60080-2 

E. cloni 10G ELITE Cells (>2 × 10 10 cfu/µg) 60052-0 

60051-1 

60051-2 

60052-1 

60052-2 

60052-3 

60052-4 

E. cloni 10G CLASSIC Cells (>5 × 10 9 cfu/µg) 60117-1 

60117-2 

E. cloni 10GF´ ELITE Cells (≥2 × 10 10 cfu/µg) 60061-1 

60061-2 

Chemically Competent Cells 

E. cloni 5-alpha Cells 

(>1 × 10 8 cfu/µg) 

E. cloni 10G Chemically Competent Cells 

(>1 × 10 9 cfu/µg) 

E. cloni 96-well 10G Chemically Competent Cells 

Cells (>1 × 10 8 cfu/µg) 

E. cloni 10G Chemically Competent Cells 

Subcloning Grade (>1 × 10 6 cfu/µg) 

E. cloni 10GF´ Chemically Competent Cells 

(>5 × 10 8 cfu/µg) 

60602-1 

60602-2 

60107-0 

60106-1 

60106-2 

60106-3 

60107-1 

60107-2 

60107-3 

60107-4 

60096-1 

60096-4 

60108-0 

60108-1 

60108-2 

60062-1 

60062-2 

YT Agar Powder 60025-1 5 packets $40 

Recovery Medium 80026-1 8 × 12 ml 

Competent Cells include Control DNA, and Recovery Medium, and are packaged as indicated: SOLOs (1 transformation per tube), 

DUOs (2 transformations per tube), FIVERs (5 transformations per tube), SixPacks (6 transformations per tube), Subcloning 

Grade (12 transformations per tube), or microplates as indicated. Recovery Medium is also available separately. The specified 

transformation efficiencies below are with pUC DNA, unless indicated otherwise. One free sample per lab. See page 7 for additional 

information. 

PLEASE NOTE: Bulk quantities (10 times larger than the largest retail package size below) and/or custom packaging are available at 

very attractive prices for all Lucigen Competent Cells. Custom packaging options include: pre-dispensed cells in standard 96-well 

plates and tubes. Please contact Lucigen. 

E. cloni 10G: F- mcrA Δ(mrr-hsdRMS-mcrBC) endA1 recA1 Φ80dlacZΔM15 Δlac×74 araD139 Δ(ara,leu)7697 galU 

galK rpsL nupGλ- tonA 

E. cloni 10GF´: [F´ pro A+B+ lacIqZΔM15::Tn10 (TetR)] /mcrA Δ(mrr-hsdRMS-mcrBC) endA1 recA1 

Φ80dlacZΔM15 Δlac×74 araD139 Δ(ara, leu)7697 galU galK rpsL nupGλ tonA 


12 reactions (SOLOs) 


12 reactions (DUOs) 


4 reactions 





24 reactions (Six Packs) 
















1 plate 

4 plates 

12 reactions 

48 reactions 

96 reactions 



Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 




43 


3


3 

GENOYTPE 

44 Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

E. cloni® 5-alpha Chemically Competent Cells 

• Direct replacements for standard cloning strains (e.g., DH5α, DH10B, 

JM109, TOP10, XL1-Blue, etc.) 

• Optimized genetics for high yields: endonuclease and recombination minus, 

for blue/white screening-capable. 

Lucigen’s E. cloni competent cells share the most useful genetic elements of standard cloning strains 

like DH5α and directly replace them in cloning protocols. E. cloni chemically competent cells provide 

the performance researchers need with ease of use—only common laboratory equipment is required. 

These cells provide solutions for a wide range of applications at economical prices. 

Useful for routine cloning, subcloning, and plasmid isolation with or without blue/white screening. 

E. cloni 5-alpha: fhuA2Δ(argF-lacZ)U169 phoA glnV44 Φ80 Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17 

Custom 


Products 

E. cloni 5-alpha Cells 

(>1 × 10 8 cfu/µg) 


Competent Cells include Control DNA and Recovery Medium, and are packaged as DUOs (2 transformations 

per tube). Recovery Medium is also available separately. The specified transformation efficiencies are with pUC 

DNA, unless indicated otherwise. 

PLEASE NOTE: Bulk quantities (10 times larger than the largest retail package size below) and/or custom 

packaging are available at very attractive prices for all Lucigen Competent Cells. Please contact Lucigen. 

CUSTOM COMPETENT CELLS 

Custom Competent Cells 

• Highest transformation efficiencies available, even ≥ 4 × 10 10 cfu/µg DNA. 

• Chemically competent or electrocompetent cells. 

• Any E. coli strain made to the highest possible efficiency 

(Lucigen's or your own). 

• Dispensed at any volume. 

• Consistent quality from over 10 years of manufacturing experience. 

Applications for large or special projects: User Benefits 




• Cloning (mutagenesis, etc.) 

• Phage display 

TG1, SS320, ER2738 strains available 

• Library construction 

• Protein expression 


• Average 2-3 week turn-around 

• 4 ml minimum 

• Guaranteed performance 

• Bulk discount 

• Confidential 

Lucigen has also increased transformation efficiencies of low competency strains by 10-100 fold in just a 

few days. Contact Lucigen for details. 

Cat. No. Size 

60602-1 

60602-2 


24 reactions (DUOs)

PHAGE DISPLAY LIBRARY APPLICATIONS 

Phage Display Competent Cells 

• Highest transformation efficiencies (≥ 4 × 10 10 cfu/µg and 

greater) available. 

• Only source for electrocompetent SS320 and ER2738 strains. 

• ER2738 strain recommended for use with Ph.D. Phage Display Libraries. 

• Create larger libraries; speed discovery. 

• Suitable for M13 phage work and protein expression. 

• Bulk cells available with custom dispensing. 

TG1 Electrocompetent Cells. An amber suppressor strain (supE) prepared as highly efficient (≥4 × 

10 10 cfu/µg) electrocompetent cells for phage display library screening. These cells are used for phage 

display and protein expression. 

SS320 Electrocompetent Cells. A non-amber suppressor strain (sometimes called MC1061F’) pre- 

pared as highly efficient (≥4 × 10 10 cfu/µg) electrocompetent cells for phage display library screening. 

These cells have the highest available transformation efficiency of any strain used for phage display. 

ER2738 Electrocompetent Cells. An amber suppressor strain (glnV) prepared as highly efficient 

(≥2 × 10 10 cfu/µg) electrocompetent cells for phage display library screening. This strain is recom- 

mended for use with New England Biolab’s Ph.D. Phage Display Kits. 

GENOYTPES 

TG1: [F' traD36 proAB lacIqZ ΔM15] supE thi-1 Δ(lac-proAB) Δ(mcrB-hsdSM)5(rK - mK -) 

SS320 (MC1061F'): [F'proAB+lacIqlacZΔM15 Tn10 (tetr)] hsdR mcrB araD139 Δ(araABC-leu)7679ΔlacX74 

galUgalK rpsL thi 

ER2738: [F'proA+B+ lacIq Δ(lacZ)M15 zzf::Tn10 (tetr)] fhuA2 glnVΔ(lac-proAB) thi-1Δ(hsdS-mcrB)5 

Products 

TG1 Electrocompetent Cells 60502-1 

60502-2 

SS320 (MC1061 F') ElectrocompetentCells 60512-1 

60512-2 

ER2738 Electrocompetent Cells 60522-1 

60522-2 

Phage Display Electrocompetent Cells 

Four transformations of each strain 


Cat. No. Size 


12 rxns (DUOs) 






60500-0 12 rxns 

Recovery Medium 80026-1 8 x 12 ml 

Competent Cells include Control DNA and Recovery Medium, and are packaged as DUOs (2 transformations 

per tube). Recovery Medium is also available separately. The specified transformation efficiencies are with 

pUC DNA, unless indicated otherwise. One free sample per lab. See page 7 for additional information. 



FREE SAMPLE 


Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

Transformation e�ciency (cfu/μg) 

4 × 10 10 

3 × 10 10 

2 × 10 10 

1 × 10 10 

0 

TG1 SS320 ER2738 TG1 

Lucigen Competitor S 

Figure 2. Transformation efficiency of Lucigen’s electrocompetent 

bacterial cells for phage display compared 

to competitor’s specification. 




45 


3


3 Cat. 

SIZE DEFINITIONS 

SOLOs: 1 transformation per tube 

DUOs: 2 transformations per tube 

FIVERs: 5 transformations per tube 

SixPacks: 6 transformations per tube 

VOLUME PER REACTION 

ElectroComp: 25 µl per reaction 

ChemComp: 40 µl per reaction 

≥1.2 × 10 10 

≥1 × 10 10 

≥0.8 × 10 9 

≥0.6 × 10 9 

≥0.4 × 10 9 

≥0.2 × 10 9 

0 

Lucigen Endura Electro 

(24 rxn) 

E�ciency (cfu/µg) 

Invitrogen Stbl4 

(25 rxn) 

Agilent SURE Electro 

(10 rxn) 

46 Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

LARGE OR DIFFICULT FRAGMENT CLONING 

NEW Endura Competent Cells 

• Clone unstable sequences with reliable results 

• Direct replacement for Invitrogen Stbl3 and Agilent SURE 

strain 

• Available as chemically competent or electrocompetent cells 

• Your choice of two packaging formats 

• High efficiency: over 1 × 10 8 cfu/µg (chem) or 1 × 10 10 cfu/µg (electro) 

• Excellent value 

Conserve clones that contain unstable sequences! 

Maintain unstable DNA. Lucigen’s Endura Competent Cells are a commonly used strain for cloning 

sequences that suffer unwanted recombination events in other strains. Clones with inverted repeats or 

other sequnces prone to recombination are commonly found in retroviral genes, and require cells such 

as Endura to be propagated stably. 

Excellent value and efficiency! Switch to Endura cells today and experience a higher level of efficiency 

and a savings in your cloning budget. 

Chemical or Electrocompetent. Whichever method of transformation you prefer, Lucigen can give you 

better efficiency and/or better prices. 

GENOYTPES 

recA13 supE44 ara-14 galK2 lacY1 proA2 rpsL20(Str R ) xyl-5 λ– leu mtl-1 F– mcrB mrr hsdS20(r B –, mB–) 

Products 

Endura Chemically Competent Cells 60240-0 

60240-1 

60240-2 

60241-1 

60241-2 

Endura ElectroCompetent Cells 60242-0 

60242-1 

60242-2 


Endura Competent Cells include Control DNA and Recovery Medium, and are packaged as SOLOs (1 

transformation per tube) or DUOs (2 transformations per tube) as indicated. Endura Electrocompetent cells 

are provided in DUOs format only. Recovery Medium is also available separately. The specified transformation 

efficiencies are with pUC DNA, unless indicated otherwise. 







No. Size 

4 rxn (DUO) 

12 rxn (DUO) 

24 rxn (DUO) 

12 rxn (SOLO) 

24 rxn (SOLO) 

4 rxn (DUO) 

12 rxn (DUO) 

24 rxn (DUO) 

FREE SAMPLE 

lucigen.com/samples

LARGE OR DIFFICULT FRAGMENT CLONING 

BAC-Optimized Replicator 2.0 & 

10G BAC-Optimized Electrocompetent Cells 

• Cells that enable the most challenging library construction and screening. 

• Highest available transformation efficiencies for library construction and 

screening. 

• Optimized for high yields of DNA. 

Lucigen offers two bacterial strains designed for BAC library construction or transformation of large 

constructs: 

BAC-Optimized Replicator v2.0 Electrocompetent Cells (≥ 2 × 10 10 cfu/µg pKanR DNA). These 

cells are required for on-demand, copy number amplification of the pSMART® BAC or pEZ BAC 

vectors in the CopyRight® Kits, thereby increasing DNA yields. BAC-Optimized Replicator Cells are 

components of Lucigen’s CopyRight Cloning Kits. 

E. cloni 10G BAC-Optimized Electrocompetent Cells (> 1 × 10 7 cfu/µg 150 kb BAC DNA; ≥ 1 × 10 10 

cfu/µg pUC DNA). The first competent cells developed by Lucigen specifically for large DNA cloning. 

Manufactured using a novel procedure that maximizes transformation efficiencies with very large inserts, 

10G BAC-Optimized Cells are an excellent choice for BAC library construction and large construct 

transformation. 

GENOYTPES 

Replicator v2.0: F- mcrA Δ(mrr-hsdRMS-mcrBC) endA1 recA1 Φ80dlacZΔM15 ΔlacX74 araD139 Δ(ara,leu)7697 galU galK rpsL 

nupG (attL araC-PBAD-trfA250 bla attR) λ-. Note that Replicator v2.0 cells are AmpR (bla gene). 

E. cloni 10G BAC-Optimized: F´ pro A+B+ lacIqZΔM15::Tn10 (TetR)] /mcrA Δ(mrr-hsdRMS-mcrBC) endA1 recA1 

Φ80dlacZΔM15 ΔlacX74 araD139 Δ(ara, leu)7697 galU galK rpsL nupGλ tonA 

Products 

E. cloni 10G BAC-Optimized Cells (≥1 × 10 10 

cfu/µg) (≥1 × 10 7 cfu/µg 150 kb BAC) 

BAC-Optimized Replicator v2.0 Cells 

(≥2 × 10 10 cfu/µg) 


Competent Cells include Control DNA and Recovery Medium and are packaged as SOLOs (1 transformation per 

tube). Recovery Medium is also available separately. The specified transformation efficiencies are with pUC DNA, 

unless indicated otherwise. 



See p. 32 for BigEasy® TSA 


Cat. No. Size 

60215-1 

60215-2 

60210-1 

60210-2 


10 reactions (FIVERs) 

25 reactions (FIVERs) 



Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 




47 


3

SECTION 4: 


Overview ..........................................50 

Cloning & Expression Systems: 

Expressioneering Technology .....51 

Competent Cells for 

Protein Expression ...........................58 


Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

Solutions 




49


4 

OVERVIEW 


Simplify your cloning and protein expression work with Lucigen’s complete cloning and expression systems. We also offer highest efficiency competent cells 

for routine cloning and expression as well as competent cells that can express even difficult or toxic proteins. 

NEW Expressioneering Technology 

Your path to surprisingly simple protein expression! 

• Enzyme-free directional PCR cloning in seconds 

• Save days of effort with ready-to-use vectors and cells – no ligation steps 

• Bacterial and Mammalian Expression systems available 

E. coli Expression 

• Your choice of T7 or Rhamnose Promoter 

• Tightly controlled expression 

• N- or C-terminal 6X His-tagged proteins 

• Enhanced solubility with cleavable 

SUMO tag 

Mammalian Expression 

• CMV promoter for robust expression in multiple cell lines 

• Smallest mammalian expression vector available 

• HA tag for easy protein purification & immuno detection 

Watch how 

it works ↑ 

50 Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

View the Expressioneering 

Technology video at: 

lucigen.com/expressovideo 

COMPETENT CELLS FOR PROTEIN EXPRESSION 





See also Competent Cells: 

✓ Phage Display p. 45 

OverExpress C41(DE3) and C43(DE3) Competent Cells. Express difficult or even toxic proteins using any E. coli T7 vector. Available as chemically competent or 


E. cloni EXPRESS BL21(DE3) Competent Cells. For routine protein expression using any E. coli T7 expression vector. Available as chemically competent or high efficiency 


Phage Display Electrocompetent Cells 

With the highest available transformation efficiency available (≥4 × 10 10 cfu/µg DNA), Lucigen’s SS320, TG1, and ER2738 Electrocompetent Cells are the cells of choice for 

phage display protein expression. See Section 4, Competent Cells, p. 43. 

Figure 1. Expressioneering Technology 

SV40 

prom/ori

CLONING & EXPRESSION SYSTEMS: EXPRESSIONEERING TECHNOLOGY 

Expresso® Cloning & Expression Systems: E. coli Expression 

• Enzyme-free directional PCR cloning in seconds! 

• Save days of effort with ready-to-use vectors and competent cells - 

NO ligation step. 

• Tightly-controlled expression of N- or C-Terminal 6xHis tagged proteins. 

• Available with 2 promoter options: T7 and tunable 

Rhamnose. 

• Enhanced solubility with cleavable SUMO solubility tag. 

Expresso Cloning and Expression Kits Selection Guide 

Expresso 

T7 

Expresso 

Rhamnose 

Expresso SUMO Solubility Tag (optional) ✓ ✓ 

Enzyme-free cloning of PCR products ✓ ✓ 

6xHis tag N-terminal or C-terminal ✓ ✓ 

Single host strain for cloning & expression � ✓ 

Tunable induction Rhamnose promoter � ✓ 

Auto Induction Reagents � ✓ 

T7 promoter for maximal expression 

levels 

✓ � 

High-level expression with Expresso T7 kits 

The Expresso T7 Cloning and Expression Systems feature the popular bacteriophage T7 promoter 

for routine high-level expression of target proteins (Figs. 1 & 2). Expression from the T7 promoter 

requires a host strain harboring the T7 RNA polymerase. The Expresso T7 Systems include two host 

strains: HI-Control 10G Competent Cells for cloning, and HI-Control BL21(DE3) Competent Cells for 

expression from the T7 promoter. The HI-Control strains feature an overexpressed lacI gene encoding 

the lac repressor for enhanced control over leaky T7 expression. The high transformation efficiency of 

HI-Control 10G Chemically Competent Cells ensures recovery of clones with precise junctions and the 

correct orientation. For most genes, > 90% of colonies will have the target gene inserted in the correct 

orientation. (Fig. 2). Verified plasmids are transformed into HI-Control BL21(DE3) cells for expression 

from the T7 promoter. Expression is inducible by IPTG or lactose (Fig. 3). 

Figure 2. Pre-processed pETite C-His vector was mixed with 1 µl of unpurified 

PCR product and transformed into HI-Control 10G Chemically Competent 

Cells. Colony PCR was performed on 18 randomly chosen colonies; 17 

of 18 contained insert of the correct size. 


Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

Figure 3. Proteins purified from Expresso T7 Vectors. 

Cellulase genes were cloned into pETite vecotrs with 

N-terminal 6xHis tags. Clones were transformed into 

Hi-Control BL21(DE3) cells for expression. Cultures 

were grown in LB SOY and induced overnight with 1mM 

IPTG. Cells were pelleted, lysed, purfied and 2 µg were 

analyzed by gel electrophoresis. The gel was stained 

with Coomassie blue. 




51 


4


Expresso vs. pET 

Comparable expression of various proteins. 

Expresso pET28a Expresso pET28a Expresso pET28a 

M - + - + - + - + - + - + M 

N-His 

DNA Polymerase 

(toxic protein) 

N-His 

BFP 

(kit control) 

Expression and purification of active 

4soluble fluorescent protein. 

52 Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

C-His 

ATP synthase 

b subunit 

(membrane 

protein) 

Figure 4. Comparable expression of various proteins. 

Genes encoding a DNA polymerase, a blue fluorescent 

protein (BFP), or ATP synthase b subunit were cloned 

into pET28a or pETite vectors with N-terminal or 

C-terminal 6x His tags. pET28a clones were transformed 

into standard BL21(DE3) cells, and pETite clones 

were transformed into HI-Control BL21(DE3) cells for 

expression. Cultures were grown in LB at 37° to an 

OD 600 of 0.6 (odd-numbered lanes) and induced for 3 

hours with 1 mM IPTG (even-numbered lanes). Cells 

were pelleted and lysed directly in SDS-PAGE loading 

buffer, and 0.05 OD equivalents were analyzed by gel 

electrophoresis. The gel was stained with Coomassie blue. 

FT W E1 E2 E3 E4 E5 E6 E7 E8 

1 2 3 4 5 6 7 8 9 10 11 12 

Figure 5. Purification of His-tagged proteins: 

HI-Control BL21(DE3) cells harboring pETite C-His 

vector containing a yellow fluorescent protein gene 

were grown at 37°C in LB media to an OD 600 of 0.6 (lane 

1), then induced with 1 mM IPTG for 4 hours (lane 2). 

Cells were harvested and lysed by sonication. Cleared 

lysate was loaded onto an Ni-NTA Sepharose® column. 

Column flow-through (lane 3, FT) and wash (lane 4, W) 

were collected. The bound YFP was eluted with buffer 

containing 300 mM imidazole (lanes 5-12, E1-E8). 


HI-Control BL21(DE3) Cells Control Leaky Protein Expression 

HI-Control BL21(DE3) Cells contain high levels of lac repressor to maintain tight control over expression 

of T7 RNA polymerases, which provides tighter control means better tolerance of potentially toxic gene 

products (Figure 4). 

Purification of Active Soluble Fluorescent Protein Using 6xHis Tag 

The N- or C-terminal 6xHis tagged proteins expressed using the Expresso T7 Cloning and Expression 

System can be rapidly affinity-purified over commercially available Nickel resins. 

Enhanced solubility with cleavable SUMO solubility tag. 

For proteins that are difficult to express in soluble form, the new pETite and pRham N-His SUMO 

vectors allow expression of target proteins with an amino-terminal 6xHis-SUMO fusion tag. SUMO 

(small ubiquitin-like modifier) is a relatively small polypeptide (100-residues) that has been shown to 

enhance the soluble expression of many proteins that are otherwise difficult to produce in E. coli. The 

6xHis-SUMO tag is removable by cleavage with SUMO Express Protease, which cleaves precisely at the 

junction between the SUMO tag and the target protein. There is no off-target cleavage, and no residues 

are left attached to the target protein after cleavage. Both the SUMO Express Protease, which is 6xHis 

tagged, and the cleaved N-6xHis-SUMO tag can then be separated from the released target protein by 

subtractive metal affinity chromatography. 

Rescue insoluble protein with SUMO protein tag 

We have used the Expresso T7 and Expresso T7 SUMO Cloning and Expression Systems for expression 

and purification of a variety of proteins, either individually or in a large-scale study (Figure 6). Initially, 

48 genes and were cloned into the pETite T7 C-His Vector. Approximately half these clones produced 

soluble, active hydrolase protein, while the others were expressed in an insoluble form. Five of the 

genes producing insoluble proteins were reamplified and cloned into the pETite SUMO vector. When 

expressed in HI-Control BL21(DE3) cells, the amount of soluble protein was significantly improved in 

four of the five cases (Table 1). The tag could be removed efficiently by SUMO Express Protease. 




Table 1. 

Fibrobacter 

succinogenes 

gene number 


Soluble protein yield (ug/ml) 

NO SUMO tag SUMO tag 

1425 0 0 

1765 0 10 

1793 0 17 

1994 0 17 

2201 0 20


A. 

1 

B. M - + | - + | - + | - + | - + | - + C. 

Gene 2201 Gene 2442 

C-His SUMO C-His SUMO 

M T S T S T S T S 


T=Total 

S= Soluble 

48 

D. 2201 SUMO 

tag removal 

protein 

- + C 

Figure 6. Large-scale cloning and expression with Expresso kits: (A) PCR products from 48 putative hydrolase genes 

ranging from ~1 to >3 kb. (B) Uninduced (-) and IPTG-induced (+) samples of HI-Control BL21(DE3) cells with 6 different 

genes cloned into the pETite C-His Vector. (C) Enhanced solubility of SUMO-tagged 2201 and 2442 gene 

products. Total cell extract and soluble fractions are shown. (D) Removal of 6xHis-SUMO tag from purified SUMO- 

2201 fusion protein by SUMO protease. –prot: uncleaved SUMO-2201 fusion protein after IMAC purification; +prot: 

SUMO protease-treated fusion protein; C: isolated 2201 protein after removal of 6xHis-SUMO fragment and SUMO 

protease by subtractive IMAC. 

Cleavage of SUMO protein tag 

After IMAC purification of the N-His-SUMO tagged protein, the tag can be removed precisely by 

the included SUMO Express Protease. The SUMO Express Protease recognizes the tertiary structure 

of SUMO rather than a short recognition sequence and cleaves precisely at the junction between the 

SUMO tag and the target protein, with no off-target cleavage. Both the SUMO Express Protease, which is 

6xHis tagged, and the cleaved N-His-SUMO tag can then be separated from the released target protein 

by subtractive IMAC. 

Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 




53 


4


4 

54 Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 


New Expresso Rhamnose System: 

Single-Host cloning and tunable expression 

The new Expresso Rhamnose Cloning and Expression System utilizes the rhaP BAD Promoter, which is 

inducible by rhamnose but tightly repressed in its absence. The rhaP BAD promoter is transcribed by the 

endogenous E. coli RNA polymerase; thus, the host cloning strain can also be used for protein expres- 

sion. There is no need for plasmid recovery and re-transformation, allowing protein expression days 

faster than with dual-host systems. 

Maximal protein expression from the rhaP BAD promoter is typically lower than from the T7 promoter, 

but can still reach very high levels (up to 100 mg/L ). For some proteins that are toxic or insoluble when 

overexpressed, the lower level of induction from the rhaP BAD promoter may actually produce a higher 

yield of functional protein. Furthermore, the level of induction from rhaP BAD is responsive to different 

concentrations of rhamnose (Fig. 7A), allowing optimization of induction levels for maximal soluble 

protein yield. The ability to tune the level of expression may be especially helpful for proteins that are 

toxic or insoluble when overexpressed. 

Convenient Autoinduction with Expresso Rhamnose Systems 

Transcription from the rhaP BAD promoter is subject to repression by glucose. When both glucose and 

rhamnose are present, glucose is metabolized preferentially and the rhaP BAD promoter remains inactive. 

Upon depletion of glucose, the rhaP BAD promoter is activated by rhamnose. Convenient autoinduc- 

tion protocols use a combination of glucose and rhamnose to adjust the timing of induction of protein 

expression(Fig. 7B). Solutions of rhamnose and glucose are provided with the Expresso Rhamnose kits. 

A. Tunable Induction B. Autoinduction 

M G 

0 

0.2 

0.1 

0.01 

0.005 

0.002 

0.001 





“Early” “Late” 

0 2 4 8 24|0 2 4 8 24 

% Rhamnose Time (hrs.) 

Figure 7A. Tuning recombinant protein expression levels 

with rhamnose induction. E. cloni 10G Cells were transformed 

with the pRham C-His Kan Vector containing a 

gene encoding a blue fluorescent protein (BFP). A starter 

culture was treated overnight with rhamnose (0 to 0.2% 

w/v) or 2% glucose (“G”). A Coomassie stained gel of total 

cellular protein shows tunable expression in response to 

rhamnose. 

Figure 7B. Regulated autoinduction with the Expresso 

Rhamnose System. E. cloni 10G Cells were transformed 

with the pRham C-His Kan Vector containing a gene 

encoding a blue fluorescent protein (BFP). Cultures 

were induced with 0.2% rhamnose plus either 0.05% 

glucose (early autoinduction) or 0.15% glucose (late 

autoinduction). Samples were harvested at the indicated 

time points for SDS-PAGE analysis.


Products 

Expresso® Rhamnose Cloning and Expression System, N-His 5 rxns 

10 rxns 

Expresso Rhamnose Cloning and Expression System, C-His 5 rxns 

10 rxns 

Expresso Rhamnose Cloning and Expression System, 

N/C-His Combo, 5 of each of N-His and C-His 

Expresso Rhamnose SUMO Cloning and Expression System, 

N-His 


The Expresso Rhamnose Cloning & Expression System contains pre-processed pRham N-His and/or pRham C-His Vector DNA, 

single-transformation E. cloni 10G Chemically Competent Cells (SOLOs) and the autoinduction reagents 20% Rhamnose solution 

and 15% Glucose solution. Also included are N-His and/or C-His Positive Control Insert DNAs, transformation positive control pUC 

DNA, and forward and reverse PCR primers to confirm clones. 

The Expresso Rhamnose SUMO Cloning and Expression System contains pre-processed pRham N-His SUMO Vector DNA, singletransformation 

E. cloni 10G Chemically Competent Cells (SOLOs) and 20% Rhamnosee solution. Also included are SUMO Positive 

Control C Insert DNA, transformation positive control pUC DNA, SUMO Express Protease, SUMO Cleavage Control Protein, and 

forward and reverse PCR primers to confirm clones. 

The Expresso T7 Cloning & Expression System contains pre-processed pETite® N-His and/or pETite C-His Vector DNA, HI-Control 

10G Chemically Competent Cells for cloning, and HI-Control BL21(DE3) Chemically Competent Cells for protein expression. Also 

included are N-His and/or C-His Positive Control Insert DNAs, and transformation positive control pUC DNA, and 

forward and reverse PCR primers to confirm clones. 

The Expresso T7 SUMO Cloning and Expression System contains pre-processed pETite® N-His SUMO Vector DNA, HI-Control 

10G Chemically Competent Cells for cloning, and HI Control BL21(DE3) Chemically Competent Cells for protein expression. Also 

included are SUMO Positive Control C Insert DNA, transformation positive control pUC DNA, SUMO Express Protease, SUMO 

Cleavage Control Protein, and forward and reverse PCR primers to confirm clones. 

Important Product Use Information: 

This product is the subject of U.S. Patent #6,709,861. Additional patent applications owned by Lucigen Corporation are pending. 

HI-Control BL21(DE3) products are sold under a license issued to Lucigen Corporation by Brookhaven Science Associates, LLC for noncommercial 

research purposes only. A separate license is required for any commercial use, including the use of these materials for research 

purposes or production purposes by any commercial entity. Information about commercial licenses may be obtained from the Office of 

Technology Commercialization and Partnership, Brookhaven National Laboratory, Bldg. 490-C, P. O. Box 5000, Upton, New York 11973- 

5000, telephone (631)-344-7134. In using this product, you agree that HI-Control BL21(DE3) cells or derivatives containing the cloned gene 

for T7 RNA Polymerase may not be distributed further to third parties outside of your laboratory, unless the recipient receives a copy of 

this limited label license and agrees to be bound by its terms. 

The 6xHis tag is licensed from Hoffmann-La Roche, Inc., Nutley, NJ and/or Hoffmann-LaRoche Ltd., Basel, Switzerland and is provided only 

for the use in research. Information about licenses for commercial use is available from QIAGEN GmbH, QIAGEN Strasse 1, D-40724 Hilden, 

Germany. Purification of 6xHis tagged proteins with Ni-NTA manufactured by QIAGEN is highly recommended for best performances and 

to avoid poor purification results. 

SUMO Express Protease is manufactured and supplied by LifeSensors, Inc. 


Size Cat. No. 

49011-1 

49011-2 

49012-1 

49012-2 

10 rxns 49010-1 

5 rxns 

10 rxns 

Expresso® T7 Cloning and Expression System, N-His 5 rxns 

10 rxns 

Expresso T7 Cloning and Expression System, C-His 5 rxns 

10 rxns 

Expresso T7 Cloning and Expression System, 

N/C-His Combo, 5 of each of N-His and C-His 

Expresso T7 SUMO Cloning and Expression System 5 rxns 

10 rxns 

49013-1 

49013-2 

49001-1 

49001-2 

49002-1 

49002-2 

10 rxns 49000-1 

49003-1 

49003-2 

HI-Control 10G Chemically Competent Cells (SOLOs) 12 rxns 60110-1 

HI-Control BL21(DE3) Chemically Competent Cells (SOLOs) 12 rxns 60435-1 

E. cloni® 10G Chemically Competent Cells (SOLOs) 12 rxns 60106-1 

SUMO Cleavage Control Protein 50 µg 30805-1 

SUMO Express Protease 200 U 30801-2 

Glucose Solution, 15% w/v 5 × 1.25 ml 49022-1 

Rhamnose Solution, 20% w/v 5 × 1.25 ml 49021-1 

Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 




55 


4


4 

45 

40 

35 

30 

25 

20 

15 

10 

5 

0 

Figure 8. pME-HA expression vector The CMV promoter 

drives expression of the PCR insert; amp and SV40 

promoters express the gene for kanamycin/neomycin 

resistance in bacteria and mammalian cells. The pUC 

origin gives high plasmid yields. CloneSmart® transcription 

terminators (T) prevent transcription into or out of 

the vector backbone, increasing clone stability. The HA 

affinity tag can be fused to the carboxy terminus of the 

expressed protein, if desired. 

Absorbance (405 nm) Over Background 

Expresso® CMV 

Multiplication Factor of Bgal Expression 

Over Background in CHO-K1 Cells 

Figure 9. CHO-K1 cells were transfected with pME-HA- 

Bgal plasmid at a ratio of 2:1 (µl TransIT-2020 transfection 

reagent : µg DNA). Cells were assayed for Bgal 

activity 24-hours post-transfection using the Mammalian 

B-Galactosidase Assay Kit (ThermoScientific, # 75707), 

and read at 405 nm on a plate reader. Absorbance 

values were divided by background absorbance (mocktransfected 

cells). 

56 Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 


New Expresso® CMV Cloning and Expression System 

• Fast: 5-second, directional cloning with no enzyme incubations, ligation, or vector prep. 

• Precise: "No scar" vector results in highly accurate sequences and correct proteins. 

• Small Vector: 3.4-kb vector gives high transfection efficiency & easy downstream 

manipulation. 

• Robust: CMV promoter provides strong, constitutive eukaryotic expression. 

The Expresso® CMV Cloning and Expression System combines instant, enzyme-free, directional cloning 

with a “no scar” vector for eukaryotic gene expression. This cloning system is based on Expressioneer- 

ing Technology, which uses in vivo homologous recombination to seamlessly clone PCR-amplified DNA 

into specially designed expression vectors without purification or enzymatic incubations. It’s the fastest, 

easiest mammalian expression system available. 

pME-HA Vector 

The Expresso CMV Cloning and Expression System features the pME-HA expression vector, which 

contains all the elements you need, and nothing you don't. The CMV promoter enables strong, constitu- 

tive expression in the smallest mammalian expression vector available. The 3.4kb vector supports higher 

tranfection efficiency and easier downstream manipulation. Combined with Expressioneering technol- 

ogy for instant, scar-free cloning, the result is surprisingly simple mammalian expression. 

Strong Expression 

The pME-HA vector provides robust protein expression, greater than or equal to that of the com- 

monly-used vector pcDNA 3.1. Protein expression from the GFP gene (0.8 kb) and the full length 

β-galactosidase gene (3 kb) was detected by Western blotting against the HA tag in CHO-K1 and COS- 

7 transfectants (Figure 10). β-galactosidase expression was also measured quantitatively in transfected 

CHO-K1 cells (Figure 9). 

250 

130 

100 

75 

55 

35 

25 

15 

Expression of GFP and β-gal in mammalian cells. 

Comparison of pME-HA vs pcDNA 3.1 MycHisC (Sigma) 

Figure 10. pME-HA expression vector The CMV promoter 

Primary: Mouse Anti-HA antibody (1:2000) 

drives Secondary: expression of Goat the PCR Anti-Mouse insert; amp IgG, and HRP SV40 conjugate promoters (1:5000) 

express Cells the Only: gene for represents kanamycin/neomycin non-transfected resistance cells in bacteria 

and mammalian cells. The pUC origin gives high plasmid 

yields. CHO-K1: CloneSmart® Transfection transcription with terminators Lipofectamine2000 

(T) prevent 

transcription COS7: into Transfection or out of the with vector Lipofectamine2000 

backbone, increasing 

clone stability. The HA affinity tag can be fused to the carboxy 

terminus of the expressed protein, if desired. 




CHO-K1 

pME-HA pc-DNA 

GFP 

β-gal 

GFP 

β-gal 

Cells Only 

GFP 

Immunoblot Analysis 

COS-7 

β-gal 

GFP 

pME-HA pc-DNA 

β-gal 

Cells Only 



Expression of Difficult Proteins 

The use of Expressioneering technology results in precise cloning, without the addition of unwanted 

sequence (such as restriction sites) typically present in multiple cloning sites. The minimal size of the 

pME-HA vector also contributes to enhanced cloning capability. Numerous GPCR genes have been 

cloned and expressed in mammalian cells using the Expresso CMV system. Western blotting was used 

to detect expression of the HA-tagged GPCRs (Figure 11), and functionality was confirmed by radioac- 

tive ligand binding assays (Figure 12). 

Expression of GPCRs in mammalian cells (COS-7) 

Vector: pME-HA 

TransIT2020 Lipo2000 

Immunoblot Analysis 

Primary: Mouse Anti-HA antibody (1:2000) 

Secondary: Goat Anti-Mouse IgG, HRP conjugate (1:5000) 

Cells Only: represents non-transfected cells 

COS7: Transfection with Lipofectamine2000 

Figure 11. Expression of GPCRs from the pME-HA vector. COS-7 

cells were transfected with constructs encoding turkey beta1adrenergic 

receptor (β1-AR) and human adenosine A1 receptor 

(A1A) with TransIT-2020 (Mirus Bio LLC) or Lipofectamine2000 

(Invitrogen) following manufacturer's recommendation. At 48 

hours post-transfection, whole cell lysates were fractionated by 

SDS-PAGE, transferred onto PVDF membrane, and detected with 

anti-HA antibody. 

Products 

Size Cat.No. 

Expresso® CMV Cloning and Expression System 5 rxns 

49031-1 

10 rxns 

49031-2 


The Expresso CMV Cloning & Expression System contains pre-processed pME-HA Vector DNA, singletransformation 

E. cloni 10G Chemically Competent Cells (SOLOs), and recovery medium. Also included are 

β-galactosidase positive control insert DNA, transformation positive control pUC DNA, and forward and reverse 

PCR primers to confirm clones. 


Specific Counts (cpm) 

Single Point Binding Assays 

Single Point Binding Assay for β1-AR receptor 

150000 

100000 

50000 

0 

Total Binding 

β1-AR Receptor A1A Receptor 

Non-specific Binding 

Figure 12. Radioligand binding data for GPCRs expressed form the pME-HA 

vector.Cells expressing b1-AR were analyzed for radioligand binding using 20 

nM [3H]-Dihydroalprenolol (PerkinElmer). Non-specific binding was determined 

in presence of excess unlabeled competitor, 10 μM (S)-(-)-Propanolol 

hydrochloride. Receptor bound ligand was captured on GF/B filters and 

counted. Cells expressing A1A receptor were analyzed by binding of 10 μM 

[3H]-DPCPX (PerkinElmer). Non-specific binding was determined in presence 

of excess unlabeled competitor, 10 μM 8-Cyclopentyl-1,3-dipropylxanthine. 

Bound ligand was captured on GF/B filters and counted. 

Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

Specific Counts (cpm) 

Single Point Binding Assay for A1A receptor 

80000 

60000 

40000 

20000 

0 

Total Binding 

Non-specific Binding 




57 


4


4 

Table 3. Competent Cells for Protein Expression 

Competent Cell 

OverExpress C41(DE3)/C43(DE3) 


OverExpress C41(DE3)/C43(DE3) pLysS 


OverExpress C41(DE3)/C43(DE3) 


OverExpress C41(DE3)/C43(DE3) pLysS 


E. cloni ® EXPRESS BL21(DE3) 


E. cloni EXPRESS BL21(DE3) pLysS or 

pLysE Electrocompetent 

E. cloni EXPRESS BL21(DE3) 


E. cloni EXPRESS BL21(DE3) pLysS or 

pLysE Chemically Competent 

58 Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 


Efficiency 

(cfu/µg DNA) 




Constructing expression 

libraries; 

maximum recombinant 

yields 


Routine cloning & 

expression 

Expressing toxic 

proteins 

> 1 × 10 10 ✓ ✓ ✓ 

> 1 × 10 9 ✓ ✓ ✓ 

> 1 × 10 6 � ✓ ✓ 

> 1 × 10 6 � ✓ ✓ 

> 5 × 10 9 ✓ ✓ � 

> 2 × 10 8 � ✓ * 

> 1 × 10 7 � ✓ � 

> 1 × 10 7 � ✓ * 

TG1 Electrocompetent Cells > 2 × 10 10 ✓ ✓ � 

SS320 (MC101 F') > 4 × 10 10 ✓ ✓ � 

ER2738 Electrocompetent > 2 × 10 10 ✓ ✓ � 

E. cloni 10GF´ Electrocompetent 

(see page 62) 

E. cloni 10GF´ Chemically Competent 

(see page 62) 

> 2 × 10 10 ✓ ✓ � 

> 5 × 10 8 � ✓ � 

* EXPRESS pLysS and pLysE cells are more tolerant of toxic gene expression than EXPRESS cells, but not as tolerant as OverExpress cells.


OverExpress C41(DE3) & C43(DE3) Competent Cells 

• Express genes cloned into any T7 vector with these BL21(DE3) derivatives. 

• Effective in expressing toxic & membrane proteins. 

• Cited in over 350 research articles ›90% recombinants. 

E. coli BL21(DE3) strains, like Lucigen’s E. cloni® EXPRESS Competent Cells provide reliable expression 

of many genes cloned into T7 expression vectors (e.g., pET or Lucigen’s pSMART®-cDNA vectors). 

However, in some cases expression is minimal or not detectable because the recombinant protein, when 

expressed, is deleterious or lethal to these standard BL21 strains. Examples of such toxic proteins include 

many membrane proteins, some cytoplasmic proteins, and nucleases. Unfortunately, successful expres- 

sion of one or more toxic proteins is often important to the experimental goal. 

Table 2: Comparison of OverExpress C41(DE3) and C43(DE3) cells with the parental strain BL21(DE3) in 

transformation and expression of heterologous proteins.** 

Strain 

A: Transformation 

Success Rate 

B: Expression-induced 

Toxicity 

a Transformation success corresponds to the presence of colonies on LB+ampicillin agar following transformation 

with a plasmid. 

b Expression toxicity corresponds to the absence of colonies on LB+ampicillin+IPTG agar following transformation 

with a plasmid. 

c Expressing plasmids corresponds to observation of a heterologous protein in the total cell pellet on Coomassiestained 

SDS-PAGE following growth of a colony in LB+ampicillin medium and induction with IPTG. 

** L. Dumon-Seignovert, G. Cariot, and L. Vuillard (2004). Protein Expression and Purification 37, 203-206. Data 

used with permission. 


C. Expressing 

Plasmids 

BL21(DE3) 16/26 (62%) 25/26 (96%) 14/26 (54%) 

C41(DE3) 28/28 (100%) 14/28 (50%) 24/28 (86%) 

C43(DE3) 28/28 (100%) 1/28 (4%) 23/28 (81%) 

100 

80 

60 

40 

20 

Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

0 

C41 BL21 C43 

Figure 12. Green Fluorescent Protein (top) or Red 

Fluorescent Protein (bottom) expressed from a T7 

promoter construct that was transformed into C41, 

BL21, or C43 competent cells spread on IPTG plates 

to induce protein expression. 


Success Rate a 

Post-expression 

Viability b 

Protein Expression c 

Figure 14. Comparison of OverExpress C41(DE3) and 

C43(DE3) cells with the parental strain BL21(DE3) in transformation 

and expression of heterologous proteins.** 




59 


4 

BL21(DE3) 

C41(DE3) 

C43(DE3)


4 

Which OverExpress cell 

strain should I use? 

It is difficult to predict which of 

the four OverExpress strains – 

C41(DE3), C43(DE3), C41(DE3) 

pLysS, or C43(DE3)pLysS – will 

work best in expressing a given 

protein. We recommend initially 

using the OverExpress Combo- 

Pack which contains 3 reactions 

each of the four OverExpress com- 

petent cell strains, to determine 

which one is best for your applica- 

tion. The OverExpress strains are 

available as electrocompetent or 

chemically competent cells. 

60 Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 


Lucigen’s OverExpress Electrocompetent and Chemically Competent Cells are strains that are effective 

in expressing toxic proteins from all classes of organisms, including eubacteria, yeasts, plants, viruses, 

and mammals. The effectiveness of these new strains in expressing toxic proteins has been validated in 

more than 350 publications. See lucigen.com. 

The OverExpress strains contain genetic mutations phenotypically selected for conferring tolerance to 

toxic proteins. The strain C41(DE3) was derived from BL21(DE3). This strain has at least one mutation, 

which prevents cell death associated with expression of many recombinant toxic proteins. The strain 

C43(DE3) was derived from C41(DE3) by selecting for resistance to a different toxic protein and can 

express a different set of toxic proteins to C41(DE3). Figure 13 graphically illustrates the advantages 

of the OverExpress Competent Cells as compared to standard BL21(DE3) cells, in expressing toxic 

proteins. 

Table 2 and Figure 14 summarize transformation effectiveness, tolerance of expression-induced toxic- 

ity, and protein expression for T7 expression plasmids coding for a variety of recombinant proteins. 

These results demonstrate that the OverExpress C41(DE3) and C43(DE3) strains are clearly superior to 

the parental BL21(DE3) in transformation and expression of toxic proteins. 

Products 


OverExpress C41(DE3) Cells 

(>1 × 10 10 cfu/µg) 

OverExpress C41(DE3)pLysS Cells 

(>1 × 10 9 cfu/µg) 


(>1 × 10 10 cfu/µg) 


(>1 × 10 9 cfu/µg) 

OverExpress ElectroComboPack 

(3 reactions each of the above 4 strains) 



(>1 × 10 6 cfu/µg) 


(>1 × 10 6 cfu/µg) 


(>1 × 10 6 cfu/µg) 


(>1 × 10 6 cfu/µg) 

OverExpress ChemComboPack 

(3 reactions each of the above 4 strains) 

Expression Recovery Medium 


(lactose minus) 


Each OverExpress Kit contains the indicated OverExpress Electrocompetent or Chemically Competent Cells in 

SOLO packaging (1 transformation per tube), Expression Recovery Medium (lactose minus), pUC19 Positive 

Control Plasmid, pAVD10 Verification Plasmid, and complete protocols. ComboPacks contain 3 reactions 

each of C41(DE3), C43(DE3), C41(DE)pLysS, and C43(DE3) pLysS, as either electrocompetent or chemically 

competent cells, as indicated. Expression Recovery Medium is also available separately. 24-reaction kits are 

multiples of the 12 reaction kit, 2 × 12 reactions. 





Cat. No. Size 

60341-1 

60341-2 

60343-1 

60343-2 

60345-1 

60345-2 

60347-1 

60347-2 









60350-1 12 reactions (SOLOs) 

60442-1 

60442-2 

60444-1 

60444-2 

60446-1 

60446-2 

60448-1 

60448-2 









60452-1 12 reactions (SOLOs) 

80030-1 8 × 12 ml


As in standard BL21(DE3) strains, OverExpress C41(DE3), C41(DE3)pLysS, C43(DE3), and C43 (DE3) 

pLysS are lysogens of λDE3. These strains carry a chromosomal copy of the T7 RNA Polymerase gene 

under the control of the lacUV5 promoter. These strains are suitable for production of protein from 

target genes cloned into T7-driven expression vectors. OverExpress C41(DE3), C41(DE3) pLysS, 

C43(DE3), and C43(DE3) pLysS are also deficient in the lon and ompT proteases. 

OverExpress C41(DE3)pLysS and C43(DE3)pLysS also carry a chloramphenicol-resistant plasmid 

that encodes T7 lysozyme, which is a natural inhibitor of T7 RNA polymerase. Cells containing pLysS 

produce a small amount of T7 lysozyme. These strains are used to suppress basal expression of T7 RNA 

polymerase prior to induction, thus stabilizing recombinants encoding particularly toxic proteins. 

Because there are no intrinsic antibiotic resistances (or plasmids) in either C41(DE3) or C43(DE3), the 

strains can be differentiated from each other and from BL21(DE3) by transformation with a strain verifi- 

cation vector, pAVD10. pAVD10 contains the uncF gene (encoding the beta-subunit of E. coli ATPase) 

under the control of the T7 promoter. This plasmid is lethal to BL21(DE3) and to induced C41(DE3), but 

it is tolerated by C43(DE3) regardless of induction. pAVD10 is provided with OverExpress Cells. 

Important Product Use Information 

OverExpress Cells are licensed exclusively to Lucigen Corporation by Imaxio S.A. under US Pat. 6,361,966 and 

others. Purchase of OverExpress Cells is accompanied by a non-exclusive, non-transferable license for research use 

only. Commercial use requires a separate license available from Imaxio S.A. OverExpress Cells are also sold under a 

license issued to Lucigen Corporation by Brookhaven Science Associates, LLC (BSA) for research use by noncommercial 

entities. Commercial entities require a license from BSA even for research use. Also see p.101. 

E. cloni EXPRESS 

Supplier “E” 

Supplier “P” 

Supplier “S” 

Supplier “N” 

Supplier “I” 

Larger Expression Libraries 

cfu/µg x 109 0 1 2 3 4 5 6 

Figure 15. Transformation efficiency comparison of 

commercially available BL21(DE3) competent cells. 


Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 




61 


4


4 

62 Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 


Products 


EXPRESS BL21(DE3) Cells 

(>5 × 10 9 cfu/µg) 

EXPRESS BL21(DE3) pLysS Cells 

(>2 × 10 8 cfu/µg) 

EXPRESS BL21(DE3) pLysE Cells 

(>2 × 10 8 cfu/µg) 

E. cloni EXPRESS ElectroComboPack 

(4 rxns each of the above 3 strains) 


EXPRESS BL21(DE3) Cells 

(>1 x 10 7 cfu/µg) 

EXPRESS BL21(DE3) pLysS Cells 

(>1 × 10 7 cfu/µg) 

EXPRESS BL21(DE3) pLysE Cells 

(>1 × 10 7 cfu/µg) 


Each E. cloni EXPRESS Kit contains: the indicated E. cloni EXPRESS Electrocompetent or Chemically Competent Cells 

in DUO packaging (2 transformations per tube), Expression Recovery Medium (lactose minus), pUC19 Positive 

Control Plasmid, and complete protocols. 

EXPRESS ComboPacks contain 4 reactions each of all 3 strains: EXPRESS BL21(DE3), EXPRESS BL21(DE3)pLysS, and 

EXPRESS BL21(DE3) pLysE in DUO packaging, Expression Recovery Medium (lactose minus), pUC19 Positive Control 

Plasmid, and complete protocols. 

Expression Recovery Medium (lactose minus) is also available separately. 





Cat. No. Size 

60300-1 12 reactions (DUOs) 
















E. cloni EXPRESS ChemComboPack 60430-1 12 reactions (DUOs) 


Expression Recovery Medium (lactose minus) 80026-1 8 x 12 ml

SECTION 5: 

Enzymes 

Enzymes ............................................66 

CAZymes ..........................................76 


Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

Solutions 




65

Enzymes 

5 

✓ 

See also our full line of 

PCR & Amplification Enzymes pp. 10-19 

NxGen Genomic Grade Enzymes 

Exceptional Performance for Demanding Applications. 

Lucigen’s NxGen enzymes are ideally suited to Next-Gen applications such as 

sequencing & array analysis that require effective and pure reagents. Achieve 

optimal results even in highly-sensitive applications. 

• Industry-leading value and quality. 

• 99% purity for consistency and reliability. 

• Convenient sizing for ease of use and ordering. 

Polymerases Also see pp. 10-19 for full line of PCR & Amplification Enzymes 

Powerful enzymes to provide accurate generation of nucleic acid polymers from a wide range of starting molecules. 

Polymerases Applications Proofreading 

phi29 Polymerase 

30221 p. 64 

M-MuLV Reverse 

Transcriptase 

30222 p. 64 

T7 RNA Polymerase 

30223 p. 65 

T7 DNA Polymerase 

30224 p. 65 

Terminal Transferase 

30225 p. 66 

Bst DNA Polymerase 

30028 See p. 18, 

Amplification & PCR 

DNA Polymerase I 

Large (Klenow), 

Exonuclease minus 

30095 p. 66 


30081 p. 67 

T4 DNA Polymerase, 

Exonuclease minus 

30085 p. 67 

66 Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

� 

cDNA synthesis 

▲ 

Transcription, 

Gene Expression 

Site-directed mutagenesis 

� 

3' Labeling 

3´→5´ 

Strong 

3´→5´ 

Very strong 




Strand 

Displacement 

Error 

Rate 


Thermal 

Stability 

Extend 

from RNA 

Primer 

Extend 

from 

Nick 

Very strong ✓ ✓ 

Strong 

15 × 10 -6 ✓ 

�� Very strong ✓ 

Labeling 

� 

Fill-in, 

mutagenesis 

� 

Applications: ▲ RT-PCR �Sequencing �Cloning 

3´→5´ 

Strong 

3´→5´ 

Very strong 

Molecular Biology Grade Enzymes 

Enzymes for Molecular Biology Applications. 

A collection of enzymes used to create or modify RNA/DNA samples, allowing 

you to easily perform: 

• Labeling or RT-PCR for cloning, sequencing or detection. 

• Phosphate addition or removal. 

• RNA Degradation. 

Strong 100 × 10 -6 ✓ ✓ 

low ✓ 

� low ✓

ENZYMES 

Ligases 

Enzymes that easily link together diverse or unmodified nucleic acid polymers, including nicked strands, blunt and cohesive ends, and hetero- 

duplex formation. 

Ligases Applications 

T4 DNA Ligase, 

Low Concentration 

30241 p. 68 

T4 DNA Ligase, High 

Concentration Rapid 

Kit 

30243 p. 68 

T4 RNA Ligase 

30244 p. 68 

Nucleases 

Ligate 

Cohesive 

Ends 

Ligate Blunt 

Ends 


Nicks in 

dsDNA 

DNA-RNA 

Heteroduplex 

Formation 

Cloning, 

Linker 

addition ✓ ✓ ✓ ✓ 

Cloning, 

Linker 

addition ✓ ✓ ✓ ✓ 

RNA 3' 

Labeling 

Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

ssRNA and 

dsRNA oligo 

joining 

RNA/DNA 

joining, 3' 

Labeling ✓ ✓ 

Efficiently modify your single or double-stranded DNA in any condition to obtain modifications at either end. 

Nucleases Polarity Substrate 

Lambda 

Exonuclease 

30261 p. 69 

Exonuclease I 

30262 p. 69 

Exonuclease III 

30263 p. 70 

RNAse I 

30104 p. 70 

5´→3´ 

DNA compatibility 

ssDNA, 

dsDNA 

Activity 

without 

5'-phosphate 

1 

3' Recessed 

Ends 

3' Protruding 

Ends 

Blunt 

Partial ss 

Extension 

Ends Nicks Digestsion Product 

✓ ✓ 3' 

ssDNA, 

dNMP 

3´→5´ ssDNA ✓ dNMP 3 

3´→5´ 

ssDNA, 

dsDNA ✓ ✓ 

ssRNA 

1 = Strongly prefers 5'-P, but will degrade without it. 

2 = Exo III cannot blunt a 3' extension. 

3 = Exo I cannot cleave the final bond, yielding dNMP's and a dinucleotide. 

2 

✓ 

5' 

ssDNA, 

dNMP 

Ribo- 

nucleo- 

tides 

Phosphorothioate 

cleavage 




67 

Enzymes 

5

Enzymes 

5 

ENZYMES: POLYMERASES 

phi29 DNA Polymerase 

Excellent strand displacement, fidelity and speed. 

phi29 DNA Polymerase is a highly processive DNA polymerase with a 

powerful strand displacement activity and a 3′→ 5′ proofreading exonucle- 

ase function. The enzyme is capable of up to 70,000 insertions per binding 

event. 

Specifications 

Unit Definition: 1 unit is defined as the amount of polymerase required to 

convert 0.5 pmol of dNTP’s into acid insoluble material in 10 minutes at 

30°C. 

Source: A recombinant E. coli strain carrying the phi29 DNA Polymerase 

gene from bacteriophage phi29. 

Unit Concentration 10,000 U/mL 

Purity (SDS-PAGE) >99% 

SS Exonuclease Functional 

E. coli 16S rDNA Contamination 100 U


T7 RNA Polymerase 

Reliable transcription. 

T7 RNA Polymerase catalyzes the 5′→3′ RNA synthesis from the 

T7 promoter. It is a DNA-dependent RNA polymerase cloned from the 

T7 bacteriophage, which recognizes the T7 promoter and terminator 

sequences with high specificity. 


Unit Definition: One unit is defined as the amount of enzyme that will 

incorporate 1 nmol of ATP into acid-precipitable material in 1 hour at 

37°C. 

Source: Purified from a strain of E. coli that expresses the recombinant 

T7 RNA Polymerase gene. 



SS Exonuclease 500 U

Enzymes 

5 


Terminal Transferase 

Effective end modification and labeling. 

Terminal deoxynucleotidyl Transferase (TdT) is a DNA polymerase that 

catalyzes the addition of deoxynucleotides to the 3’ hydroxyl terminus of 

ssDNA or dsDNA. 3’ tailing can be induced with the addition of 

1mM Co 2+ . This protein is sold as an N-terminal truncation of the terminal 

transferase gene attached to an N-terminal fusion tag. 


Unit Definition: 1 unit is defined as the amount of polymerase required to 

convert 1 nmol of dTTPs into acid insoluble material in 1 hour at 37°C. 

Source: An E. coli strain that carries the cloned terminal transferase gene 

from calf thymus. 





T4 DNA Polymerase, Exonuclease Minus 

Only source for recombinant T4 DNA Polymerase, 

Exonuclease Minus. 

Potential applications include enhancing DNA 

sequencing reads. 

T4 DNA Polymerase, Exonuclease Minus is a DNA-dependent DNA poly- 

merase which catalyzes the 5’→3’ synthesis of DNA and requires template 

and primer. Additionally, the enzyme lacks 3’→5’ exonuclease activity. 

Source: Purified from a strain of E. coli that carries a T4 DNA Polymerase, 

Exonuclease Minus expression plasmid. 

Applications: DNA Sequencing reaction. 

Heat inactivation: 70°C for 15 minutes. 

Storage: Enzyme and buffer should be stored at -20°C. Enzyme is stable for 

12 months if handled properly. 

Additional Information: May increase the DNA sequencing read-lengths. 

Products 

T4 DNA Polymerase, 

Exonuclease Minus 


Size Cat. No. 

300 U 30085-1 

1,500 U 30081-3 

Recombinant T4 DNA Polymerase, Exonuclease Minus is supplied at a concentration 

of 3,000U/ml in a storage buffer of 50% glycerol, 100 mM KPO 4 (pH 6.5) and 1 mM 

DTT. Also supplied is 10X DNA Polymerase Buffer A composed of 100 mM Tris-HCl 

(pH7.9), 100 mM MgCl 2 , 500 mM NaCl and 10 mM DTT. 1,500 U is supplied as 

5 × 300 U. 



Recombinant T4 DNA Polymerase. 

Many applications including creating blunt-ends and 

in vitro mutagenesis 

T4 DNA Polymerase is a DNA-dependent DNA polymerase which catalyzes 

the 5’→3’ synthesis of DNA and requires template and primer. Additionally, 

the enzyme has a strong 3’→5’ exonuclease activity. 

Source: Purified from a strain of E. coli that carries a T4 DNA polymerase 

expression plasmid. 

Applications: Fill in of 5’ overhanging ends to create blunt-ends, Exonucle- 

ase removal of 3’ overhanging to create blunt-ends, in vitro mutagenesis. 

Heat inactivation: 70°C for 15 minutes. 

Storage: Enzyme and buffer should be stored at -20°C. Enzyme is stable for 

12 months if handled properly. 

Additional Information: 

• T4 DNA Polymerase is active in many buffers and can often be used 

directly in a restriction digest reaction. 

• T4 DNA Polymerase exhibits no strand displacement activity. 

Products 


Recombinant T4 DNA Polymerase is supplied in Storage Buffer and includes 10X 

DNA Polymerase Buffer A. (See specifications above). 

Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

Size Cat. No. 

T4 DNA Polymerase 300 U 30081-1 

1,500 U 

(5 × 300 U) 

✓ 

30081-3 

Bst Polymerase 

See page 19 




71 

Enzymes 

5

Enzymes 

5 

ENZYMES: LIGASES 

T4 DNA Ligase 

Reliably join double-stranded nucleic acid chains. 

T4 DNA Ligase catalyzes joins strands of dsRNA and dsDNA by forming 

phosphodiester bonds. The enzyme efficiently joins blunt and cohesive 

ends and repairs single stranded nicks in duplex DNA, RNA, or DNA/RNA 

hybrids. 

T4 DNA Ligase 

Version 

Ligase Low 

Concentration 

High 

Concentration 

Rapid Kit 

72 Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

Size [Ligase] Provided Buffer 

100,000 U 120,000 U/mL 

150 rxn 600,000 U/mL 

10X T4 DNA Ligase 

Buffer 

2X Rapid Ligation 

Buffer, 

10X T4 DNA Ligase 

Buffer 

10X T4 DNA Ligase Buffer is composed of 500mM Tris-HCI, 100 mM 

MgCl 2 , 50 mM dithiothreitol, 10 mM ATP, pH 7.6 @ 25°C. 

2X Rapid Ligation Buffer is composed of 132mM Tris-HCI, 20 mM MgCl 2 , 

2 mM dithiothreitol, 2 mM ATP, 15% PEG, pH 7.6 @ 25°C. 


Unit Definition: 1 unit is defined as the amount of T4 DNA Ligase required 

to join 50% of 100 ng of DNA fragments with cohesive termini in 50 µl 1X 

T4 DNA Ligase Buffer following a 30 minute incubation at 23°C. 

Source: A recombinant E. coli strain carrying the cloned T4 DNA Ligase 

gene. 

SS Exonuclease 6,000 U

ENZYMES: NUCLEASES 

Lambda Exonuclease 

Modify diverse 5’ ends of dsDNA. 

Lambda Exonuclease selectively degrades the 5′-phosphorylated (but not 

5’-hydroxyl) strand of dsDNA. It is a highly processive enzyme that does not 

initiate at nicks or gaps, though it will degrade a 5′-overhanging tail from 

dsDNA non-preferentially. 


Unit Definition: One unit is defined as the amount of enzyme required to 

produce 10 nmol of acid-soluble deoxyribonucleotide from double-strand- 

ed substrate in 30 minutes at 37°C. 

Source: Purified from a strain of E. coli that overexpresses the exonuclease 

gene from bacteriophage Lambda. 



Endonuclease 1500 U

Enzymes 

5 

ENZYMES: NUCLEASES 

Exonuclease III 

Create 5’ overhangs with ease. 

Exonuclease III will digest double-stranded DNA to leave 5′ overhangs. It will 

digest dsDNA in the 3′ → 5’ direction, or digest the RNA strand of an RNA- 

DNA heteroduplex. Exonuclease III can digest from blunt or 3’-recessed ends, 

but not 3′-protruding ends. 



produce 1 nmol of acid-soluble total nucleotide in 30 minutes at 37°C. 

Source: Purified from a strain of E. coli that expresses the recombinant 

Exonuclease III gene. 



Endonuclease 1000 U

MODIFYING ENZYMES AND INHIBITORS 

RNAse Inhibitor 

Eliminate RNAse activity. 

RNAse Inhibitor is a potent inhibitor of ribonucleases such as RNase A, 

RNase B, and RNase C. The 52 kDa protein is a fusion of the porcine RNAse 

Inhibitor gene with a proprietary 22.5 kDa protein tag. 



inhibit by 50% the hydrolysis of cytidine 2',3'-cyclic monophosphate by 5 ng 

of RNAse A. 

Source: A recombinant E. coli strain carrying the porcine RNAse 

Inhibitor gene. 




Enzymes 

5 

Can't find what you need? 

Please tell us how to help! 

Contact us at lucigen@lucigen.com. 

76 Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

CARBOHYDRASES 

CAZyme Carbohydrases 

• Pure: no interfering salts, sugars or colored components. 

• Recombinant: host has no endogenous biomass-degrading activities. 

• Ready-to-use: no ammonium sulfate to remove before use. 

Carbohydrases (glycosyl hydrolases; EC 3.2.1.-) catalyze the hydrolysis of the glycosidic bond between 

two or more carbohydrates or between a carbohydrate and a non-carbohydrate moiety. These pro- 

teins are an increasingly important class of enzymes with numerous research and industrial applications. 

Information on individual carbohydrases is available on our web page, lucigen.com/store/CAZyme- 

Carbohydrases by selecting the description tab and clicking on the enzyme name. 

The advantages of Lucigen's CAZyme carbohydrases include: 

Recombinant enzymes >90% pure. 

• No unnecessary proteins, ammonium sulfate salts, sugars or colored components that can interfere 

with enzyme assays or process studies. 

• No endogenous biomass-degrading activities from host organism used for production. 

• Other available products contain mixtures of many different enzymes or can vary significantly from 

batch to batch. 

Highly-concentrated enzyme solutions. 

• Enables rapid analytical work. 

• Allows work with biomass solids without dilution from the enzyme. 

• Other available products are often supplied as ammonium sulfate precipitates. 

Extensively analyzed enzyme activity. 

• Guarantees performance. 

• Uses standard methods that can be replicated in any laboratory. 

• Eliminates confusion over potentially obscure units used in other available products. 





Products 

Clostridium thermocellum enzymes 

β-GLUCOSIDASE ACTIVITY 

Gene Locus Other Activities CAZy Family Size Cat No 

BglA Cthe_0212 GH1 2 mg 30572-1 

ENDO-CELLULASE ACTIVITY 

CelE Cthe_0797 xyloglucanase CE2 GH5 2 mg 30553-1 

CelD Cthe_0825 xyloglucanase GH9 2 mg 30554-1 

CelH Cthe_1472 xyloglucanase CBM11 GH26 GH5 2 mg 30555-1 

CelC Cthe_2807 GH5 2 mg 31552-1 

CelG Cthe_2872 GH5 2 mg 30556-1 

ENDO-MANNANASE ACTIVITY 

ManA Cthe_0032 CBM6 GH26 2 mg 30610-1 

EXO-CELLULASE ACTIVITY 

CelI Cthe_0040 GH9 CBM3 2 mg 30557-1 

CelA Cthe_0269 GH8 D1 2 mg 30558-1 

CelO Cthe_2147 CBM3 GH5 2 mg 30559-1 

CelK (reducing activity) Cthe_0412 CBM4 GH9 2 mg 30560-1 

CelR (nonreducing activity) Cthe_0578 GH9 CBM3 2 mg 30561-1 

XYLANASE ACTIVITY 

XynY Cthe_0912 CBM22 GH10 CE1 2 mg 30534-1 

XynZ Cthe_1963 GH10 2 mg 30535-1 

XynA Cthe_2972 CBM6 GH11 2 mg 30533-1 

CELLOBIOHYDROLASE ACTIVITY 

CbhA Cth_0413 

Bacillus enzymes 



GH9 CBM3 ,CBM 4_9, CelD_N 

Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

2 mg 30620-1 

Xylanase 1 Proprietary GH10 2 mg 31531-1 

Xylanase 2 Proprietary GH10 2 mg 31532-1 

β-XYLOSIDASE ACTIVITY 

Xylosidase 1 Proprietary GH43 2 mg 31511-1 

β-GLUCANASE ACTIVITY 

β-Glucanase 1 Proprietary GH10 2 mg 31591-1 

β-Glucanase 2 Proprietary GH10 2 mg 31592-1 

β-GLUCOSIDASE ACTIVITY 

β-Glucosidase 1 Proprietary GH1 2 mg 31571-1 

ARABINFURANOSIDASE ACTIVITY 

Ara 1 Proprietary Alpha-L-AF_C 2 mg 30501-1 

α-GALACTOSIDASE ACTIVITY 

NEW α-Galactosidase 1 Proprietary GH36 0.2 mg 30640-1 

β-GALACTOSIDASE ACTIVITY 

NEW β-Galactosidase 1 Proprietary GH42 0.2 mg 30650-1 

OTHER ACTIVITIES 

Expansin Proprietary GH42 2 mg 30502-1 

Dictyoglomus turgidum enzymes 

ENDO-CELLULASE ACTIVITY 

CelA** Dtur_0670 xyloglucanase 

glucomannanase 

galactomannanase 

mannanase 


GH5 2 mg 31551-1 

NEW XynA Dtur_1647 GH10 2 mg 30533-1 

Proprietary source enzymes 

α-GLUCURONIDASE ACTIVITY 

NEW α-Glucuronidase 1 Proprietary GH67 2 mg 30630-1 




77 

Enzymes 

5

SECTION 6 


Overview ......................................... 80 

Random Shear 

BAC/Fosmid Libraries .................... 81 

NextGen & Sanger Sequencing .... 85 

Other Libraries/Cloning ............... 88 

Library Screening ........................... 92 

DNA Prep ........................................ 94 


Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

Solutions 




79


6 

OVERVIEW 


Your project can benefit from our expertise in library construction, 

screening, cloning and other genomic analysis. Lucigen scientists have 

constructed hundreds of libraries including over 100 Random Shear BAC 

Libraries from metagenomes, microbes, and plant and animal species for 

clients all over the world. 

BAC/Fosmid Libraries and NextGen Sequencing 

Random Shear BAC 

• Unbiased and Gap-Free. 

• Complete Coverage with a 

Single Library. 

• Large inserts: >100kb. 

Other Libraries & Cloning 

Difficult Genome 

• Stable inserts even from A/T-rich genomes. 

• Up to 30 kb in linear pJAZZ® Vector. 

Large/Difficult Insert Cloning 

• Repetitive, G/C- or A/T-rich inserts cloned 

efficiently. 

• Up to 30 kb in linear pJAZZ® Vector. 

NanoClone & GapFree Shotgun 

• Libraries constructed with as little as 1 ng of 

target DNA. 

• Inserts stabilized in pSMART® transcriptionfree 

vectors. 

cDNA 

• Full-length, size- selected libraries from RNA, 

cells, or tissue. 

• Created in pSMART Vector or your plasmid 

of choice. 

Metagenomic 

• DNA isolated from environmental or complex 

samples. 

• BAC or fosmid libraries created from minimal 

starting material. 

80 Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

Novel Fosmid 

• Random sheared insert 

DNA eliminates bias. 

• Insert size up to 90 kb. 

Library Screening 

Pooling & Superpooling 




Complete package service, such as BAC library construction, library 

screening, genome-wide BAC end sequencing, building the BAC minimum 

tile of a genome or chromosome, position-cloning of candidate genes/ 

QTLs/transgenic loci, or complete NextGen 454 or Sanger sequencing and 

analysis are available. We can also create custom clones from otherwise 

uncloneable DNA. 

• Speed discovery and reduce screening costs. 

• Convenient. No radioactive materials 

needed. 

• Use PCR to screen pools of clones to identify 

clone(s) of interest. 

Gridding & Colony Filters 

• DNA from cells robotically printed and fixed 

onto nylon filters. 

• Rapid screen for clone(s) of interest by 

hybridization. 

Colony Preparation 

• Picking, re-arraying, duplicating. 

• Consistent assessment of colony roundedness, 

size and color. 

• Convenient arrangement in 384-well plates. 

Library Screening 

* Can be used in conjunction with other Lucigen services or as a stand-alone service. 

Partial Digestion BAC 

• Lucigen’s experts screen your library for your 

sequence of interest. 

• Confidential, cost- and time-efficient. 

• High-quality, large-insert, partial 

digestion BAC Libraries. 

• Guaranteed average insert 

size: 120kb ~ 200+kb. 

• Fast turn-around time. 


NEW NextGen Sequencing* 

• Roche 454 multiplexed data. 

• As little as 1 µg required. 

• Cost effective results. 

DNA Prep & Sequencing 

BAC end sequencing 

• Up to 700 bp per run. 

• Minimum of 90% successful reads. 

• Any scale: single read to genome-wide BAC 

end sequencing. 

BAC Shotgun Sequencing 

• Entire BAC/fosmid sequenced efficiently. 

• Chimera-free clones provided. 

BAC DNA Miniprep 

• Ultra pure HMW Genomic DNA from either 

96-well or 384-well plates. 

• DNA for gene discovery and 

BAC end sequencing. 

DNA Sequencing 

• Sanger Sequencing on ABI 3730xl 

instruments 

• NextGen Sequencing on Roche 454 platform 

HMW Genomic/BAC DNA Prep 

• Genomic DNA up to Megabase-size. 

• Prepared from any source. 

• For Next-Gen Sequencing, 

transgene construction, etc. 

• Large-scale prep for individual circular or 

linear BACs.

BAC/FOSMID LIBRARIES 

Random Shear BAC and Fosmid Libraries 

NO GAPS, even in centromeres! 

• Unbiased BAC libraries without gaps. 

• Larger inserts, fewer empty clones. 

• Generate more results, faster, for less money. 

Conventional BAC libraries are inherently biased and incomplete, due to non-random methods of DNA 

preparation and problems with conventional cloning vectors. Lucigen has developed technologies that 

overcome many of these problems, producing much more even, complete, and higher quality genomic 

libraries. 

• True, unbiased BAC libraries. Lucigen’s exclusive Random Shear Cloning Technology and 

CloneSmart® transcription-free vectors eliminate biases in DNA preparation and cloning. Clone gaps 

and sequencing gaps are practically eliminated. 

• More BAC recombinants per nanogram of input DNA. Vector preparation methods, high effi- 

ciency CopyRight® v2.0 BAC vectors, and the 10G BAC-Optimized Replicator v2.0 Competent Cells 

substantially increase recombinant yields. 

• Large BAC insert size. Random Shear BAC Libraries have a guaranteed average insert size of 100 kb 

or larger. 

• Easy production of BAC DNA. A simple, single-step copy number induction in the CopyRight v2.0 

system ensures high yields of BAC DNA, resulting in a >95% success rate in BAC end sequencing. 

• Dramatically reduced finishing costs. Lucigen’s custom Random Shear BAC Libraries offer the 

greatest value in what counts…results obtained per dollar spent. 

Problems with conventional BAC libraries 

Gaps due to partial restriction digestion 

• Uneven distribution of restriction enzyme sites results in biased libraries with numerous gaps. 

• Generating up to 40X genomic coverage in a BAC library may not eliminate this inherent 

shortcoming. 

Deleterious transcription from lacZ 

• Reliance on indicator genes such as lacZ often results in transcription/translation of the insert during 

expression of the indicator. 

• This can lead to partial deletions, sequence rearrangements, or loss of insert, especially if the insert 

expresses a deleterious protein. 

Lucigen's solutions to problems with conventional BAC libraries 

Gaps eliminated by Random Shearing 

• Lucigen’s novel methods to randomly shear HMW genomic DNA result in large fragments up to 

250kb and virtually eliminates gaps normally caused by restriction site bias (see fig. 1). 

• The combination of Random Shear technology coupled with Lucigen's DNATerminator® End Repair 

reagents, high stability CopyRight® v2.0 BAC cloning vectors, and E. cloni® 10G BAC-Optimized 

ReplicatorT v2.0 Competent Cells results in truly unbiased BAC genomic libraries with consistently 

large inserts (fig. 2 on next page). 


~Mb 

400 

300 

200 

100 

50 

kb 

Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

See lucigen.com for 

Technical Application Note 

on Random Shear Teachnology. 

Uncut 

Sau3Al 

BamHl 

EcoRi 

Hindlll 

M M 

~Mb 

400 

300 

200 

100 

50 

kb 

Figure 1. Mouse genomic DNA was digested to completion 

by several restriction enzymes (Left panel) or 

fragmented by random shearing (Right panel). Lanes: 

1, Uncut DNA; 2, Sau3A; 3, BamHI; 4, HindIII; 5, EcoRI. 

Only Sau3A digested the band at ~1 Mb. In contrast, all 

of the DNA was reduced to 100-400 kb as the degree 

of random shearing was increased. The arrow indicates 

undigestable megabase DNA. 




81 


6


6 

80 

60 

40 

20 

82 Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

0 


Figure 2. Genomic DNA was isolated from potato tissue, randomly sheared, size-selected to >100 kb, and 

cloned into the pSMART® BAC vector. DNA from minipreps was digested with NotI to excise inserts. The 

vector band is visible at 7 kb. 

Genome gaps and uneven distribution of conventional BAC clones 

0.1 3.1 6.1 9.1 12.1 15.1 18.1 21.1 24.1 27.1 29.2 

Centromere 1 

F2J7 

F15O4 

F14D7 

Core 

F14G11 

F20D23 

F4F7 

T13M22 F28G4 T24F19 F13P3 

Even distribution of Random Shear BAC Clones 

Lucigen is the only source of custom BAC libraries that are truly representative 

of the input genomic DNA 

• No gaps or missing sequences. 

• Finishing costs are dramatically reduced, genomic analyses are completed faster, and critical genes 

are obtained with much less work and expense (see Table 1). 




M Notl-cut random shearedBAC clones M 

The Power of Random Shear BAC Technology 

• Clone coverage is uniform across all the probed regions, including the centromeric regions, in our 

Random Shear Libraries (see Fig 3). 

• Lucigen’s Random Shear BAC Libraries can close existing centromeric gaps even in "finished" physi- 

cal and sequence genomic maps. 


Lucigen Random Shear Clone 

Known genomic sequence 

Overgo Oligonucleotide probe 

Genomic gaps 

F5E12 

F9A12 

F25O15 

Figure 3. Closing gaps in the Arabidopsis genome with a Random Shear BAC Library. Clone coverage was uniform across all the probed regions, including 

the centromeric region, in the Random Shear Library (red horizontal bars). In contrast, these regions show vastly different representation (black vertical bars) 

in the Arabidopsis genome project using restriction digest technology (=1, 15, 75, or =1 clone per 0.1 Mb for each region probed, blue lines from left to right; 

17X coverage overall). 

kb 

150 

100 

50 

Vector


Table 1. Comparison of finishing costs for an average BAC library 

BAC library Character 

Random Shear 

Partial 

Digestion 

What you get: 

Unbiased; 

No gaps 

Biased; 

gaps 

Needed Libraries 

(coverage) 

1 (10X) 

• Unbiased Random Shear BAC libraries in 384-well plates 

• Additional optional tools available - high density colony filters, BAC DNA pools, BAC mini-prep in 

96-well plates, BAC end sequences and more. 

Finishing 

cost 

($US) * 

Up to $1 

million 

≥ 2 (>20X) ≥ $2 million 

* Finishing cost includes BAC end sequencing, whole genome physical mapping, and integrating the physical map with 

about 1000 genetic markers. 


Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

See lucigen.com/services for 

Partial Digestion BAC Libraries 




83 


6


6 

Figure 4. Random Shear Fosmid Libraries with example 

average insert sizes: 90~100 and ~50 kb. 

84 Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 


M Random shear Fosmid clones (average insert size 90~100kb) M 

M Random shear Fosmid clones (average insert size ~50kb) M 

Custom Fosmid Libraries 

No Size Limitation due to Packaging, Truly Unbiased Libraries 

• Unbiased fosmid libraries. 

• Larger inserts, fewer empty clones. 

• Generate more results in less time with lower costs. 

Lucigen offers Custom Fosmid Library Construction Services featuring our exclusive Random Shear 

technology and the high efficiency CopyRight® v2.0 pSMART FOS vector. 




Random Shear Fosmid Libraries 

• True, unbiased Fosmid libraries. Lucigen's exclusive Random Shear 

Cloning Technology and CloneSmart® transcription-free vectors eliminate 

biases in DNA preparation and cloning. Clone gaps and sequencing gaps 

are practically eliminated. Lucigen's technology eliminates the potential 

problem of generating chimeric clones-a common problem with conven- 

tional fosmid methods. 

• Largest Fosmid insert sizes available. The average insert size of Lucigen 

Fosmid Libraries is up to 90 kb with a minimal (~10 kb) window of variation 

(Figure 4). Conventional fosmid libraries have a limitation of an average 

insert size 40 kb. 

• More Fosmid recombinants per nanogram of input DNA. Lucigen's 

vector preparation methods, high efficiency CopyRight v2.0 FOS vectors, 

and the E. cloni® FOS Replicator Cells substantially increase recombinant 

yields. Conventional fosmid cloning requires high concentration and mi- 

crogram quantities of purified large-insert DNA for ligation and packaging. 

Lucigen's technologies use 100-fold less DNA. 

• Easy production of Fosmid DNA. A simple, single-step copy number induction in the CopyRight 

v2.0 system ensures high yields of Fosmid DNA, resulting in a >95% success rate in Fosmid end- 

sequencing. 

Kb 

What you get: 

• Unbiased Random Shear Fosmid libraries without arraying (bulk glycerol stock) or arraying in 

384-well plates. 

100 

50 

Kb 

100 

50 

• Additional optional tools available - high density colony filters, Fosmid DNA pools, Fosmid mini- 

prep in 96-well plates, Fosmid end sequences and more. 

Partial Digestion BAC Libraries 

• High-quality, conventional BAC library 

• Economical alternative to Random Shear method 

Lucigen can construct a typical 5~10X BAC partial digestion library using one or more of the following 

restriction enzymes: HindIII, EcoRI, BamHI. Additional coverage and/or additional enzyme digestion 

options are available. 

What you get: 

• BAC libraries as frozen clones in 96- or 384-well plates 

• Shipped with barcode labels on lids and sides in Lucigen’s library storage boxes 

• A library construction report, technical support, and guidelines for safe handling of your custom 

BAC library are also provided 


NEXTGEN & SANGER SEQUENCING 

NextGen Sequencing Services 

Lucigen is the recognized leader in creating DNA libraries from your difficult-to-clone genomic 

samples. Now, you can also get high-quality NextGen Sequencing data with fewer gaps from the same 

company that has successfully made over 1,000 libraries. 

High-quality, gap-free NextGen Sequencing data 

• Unbiased multiplex BAC and viral libraries – fewer gaps 

• Superb accuracy on a small scale – as little as 1 µg input required 

• Roche 454 multiplexed NGS data – outstanding read length and coverage 

• Cost effective results – our experience delivers superior value 

Coverage from Lucigen library 

291 

0 

Contigs from competitor library 

Lucigen’s clone-free library enabled a single assembled contig (top panel – CLC 

Genomics Workbench so�ware used) as compared to a competitor’s library that 

le� numerous data gaps from the same starting material (lower panel – DNA Star 

so�ware used). 

Also available: 

Sanger Sequencing Services 

High throughput (96-well) library sequencing, using ABI 3730xl sequencers, offers an economical 

and efficient means to expedite your research. Whether in conjunction with Lucigen’s Custom Library 

Construction Service or performed on other samples, Lucigen’s Custom Sequencing will provide highquality 

results with a fast turn around time. Typical sequencing reactions yield >700bp with a >90% 

success rate. A confidentiality agreement ensures the complete security of all client information, project 

data, and sample information. 


Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 




85 


6


6 

86 Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 


BAC Shotgun Sequencing 

• Complete BAC sequence provided quickly (1~2 weeks). 

• Library of clones provided at no additional cost. 

• All information kept confidential. 

Lucigen offers full length insert sequencing of large-insert cosmid, fosmid, and BAC clones using 

shotgun subcloning. Purified DNA from the cosmid, fosmid or BAC clone is randomly sheared, end- 

polished, size-selected, and subcloned into Lucigen’s exclusive pSMART® Vector, and then transformed 

into E. cloni® 10G Competent Cells. The colonies of the shotgun library are robotically picked into 

96-well plates for high-throughtput DNA preparation and Sanger sequencing. 

Our service includes: 

• Large-insert DNA preparation (optional), shotgun subcloning, and library construction 

• Colony picking, overnight culturing, and DNA preparation from clones 

• DNA sequencing at any scale from a single sample to a 96-well high-throughput processing. 

Service Features 

• Exclusive pSMART shotgun library construction 

• pSMART GC or triple-A vector capable of cloning inserts up to 10kb (typically 2-4kb) 

• 95% or higher frequency of clones with inserts 

• No chimeras 

• Construction of libraries in custom plasmid vector available 

• Fast turnaround time (1~2 weeks) 

What you get: 

• Your DNA sequences are transmitted via secure internet connection or provided on appropriate 

computer media. 

• We will provide the complete shotgun library at no additional cost. 






BAC End Sequencing Services 

• Over 90% sequence success rate. 

• Over 700bp per read. 

• All information kept confidential. 

Lucigen offers BAC end sequencing with universal primers to the BAC vector, or gene-specific primers 

(supplied by the client). Our service includes robotic colony picking, overnight bacterial cell culture, 

DNA extraction from clones, DNA sequencing at any scale from a single sample to 96- or 384-well 

high-throughput processing 

Service Features: 

• Robotic colony picking and overnight culturing. 

• Automated high-throughput DNA preparation. 

• BAC end DNA sequencing with high success and long reads. 

Typical results for Lucigen’s BAC end sequencing service provide our clients with an average of at least 

90% success and reads >700bp for both forward and reverse directions (Figure 5). 

What you get: 

• Your DNA sequences are transmitted via secure internet connection or provided on appropriate 

computer media. 

• We provide raw sequence data and/or auto-trimmed BAC end sequences in FAST/TX formats. 

Figure 5. Example of a BAC end sequence with read length over 800 bp. 


Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 




87 


6


6 

“TIGR had tried several times [to 

make a large-insert Tetrahymena 

library], but the largest size range 

we could obtain was 4-6 kb. Lucigen 

was able to provide a library in the 

M 1 2 3 4 5 6 7 8 9 10 M 

pJAZZ® vector with an average insert 

size of 12 kb and some inserts as 

large as 20 kb.” 

-- Dr. Robert Coyne, 

The Institute for Genome Research 

(TIGR) 

Fco Fco -pJAZZ 3-6 3-6 kb 

M 1 2 3 4 5 6 7 8 9 10 M 

Figure 6. F. columnare genomic libraries (~70% AT) constructed 

in the pJAZZ vector. Plasmids were digested with 

NotI. M =1 kb ladder. The 2.2 kb and 11.8 kb bands are the 

vector arms of pJAZZ. 

-Data courtesy of A. Karsi and M.L. Lawrence, Mississippi State Univ. 

Contigs 

1400 

1200 

1000 

800 

600 

400 

200 

0 

Fco -pJAZZ 6-10 kb 

0 1 2 3 4 5 6 7 8 

Coverage 

88 Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

12 kb 

4 kb 

2 kb 

12 kb 

4 kb 

2 kb 

Figure 7. The F. columnare genome in the pJAZZ vector was 

sequenced to seven-fold coverage. Only 10 clone gaps were 

found in the 3.2 MB genome. 

OTHER LIBRARIES/CLONING 

Large or Difficult Insert Cloning Service 

Lucigen’s scientists have developed reagents and methods that enable the cloning 

of any DNA fragment—even “unclonable” inserts. Using proprietary methods our 

scientists can clone any size fragment. Features such as CloneSmart transcription 

terminators stabilize inserts, and a wide array of vectors can be used to ensure 

success. 

• Choice of linear pJAZZ vector, pSMART plasmid, or BAC vectors to obtain 

positive results. 

• Highly repetitive, A/T-, or G/C-rich inserts can be stably cloned. 

• Any size insert, up to 100kb+, can be cloned in Lucigen’s vectors. 

You provide the DNA insert and Lucigen provides the tools and expertise to clone it. 

What you get: 

• Receive your positive clone(s) in glycerol stock. 

• A complete report is provided, including data from quality testing and any sequence data obtained. 

bSMART Large Insert (10-20 kb) Libraries 

Exclusive Linear Vector technology gets every gene and sequence 

• Libraries created from difficult genomes such as Flavobacterium columnare 

(69% A/T). 

• Inserts up to 30kb in the linear pJAZZ® Vector. 

Lucigen’s first-of-its-kind BigEasy® pJAZZ® linear cloning vectors represent a major advance in large in- 

sert library construction. Circular plasmid vectors become supercoiled in the host cell, causing torsional 

stress and insert instability. In contrast, pJAZZ vectors remain linear and torsion-free. 

• Sequences that are unclonable in plasmid vectors are easily and stably cloned in pJAZZ. 

• Superior insert stability allows the construction of more accurate and complete libraries, from


Shotgun Libraries 

• Libraries created from precious samples. 

• No deletions or missing clones. 

Many laboratories worldwide depend on Lucigen's NanoClone® and GapFree services for economi- 

cal construction of high accuracy, unbiased shotgun libraries. In fact, Lucigen has become the leading 

provider of custom shotgun libraries, particularly with “difficult” DNA. Lucigen scientists have discovered 

numerous novel genes from phage and viral metagenomic DNA isolated directly from boiling hot springs. 

NanoClone Shotgun Libraries 

• Access entirely new genomes with Lucigen's NanoClone technology! 

• Send us as little as 1 ng of target DNA - Lucigen will generate a library of 

>1 × 10 5 plasmid clones. 

Trace amounts of gel-separated chromosomes, genomic DNA, uncultured environmental microbes, or 

base-modified phage are sufficient to generate large plasmid libraries (see Table 2). 

Many of these samples yield few or no clones using conventional techniques, which often require 5-10 µg 

of starting material. 

What you get: 

• Size-specific, random physical shearing (HydroShear fragmentation). 

• High efficiency cloning into a pSMART® or other Lucigen vector. 

• QC analysis to confirm size and sequence identity of randomly sampled clones, as well as overall 

cloning efficiency. 

Table 2. Examples of genomic libraries prepared using the NanoClone technology 

Sample 

Amount of 

Starting DNA CFUs/Library Insert Sizes Background 

Cultured cyanophage 1 ng 1 × 10 5 1-2 kb < 1% 

Freshwater environmental phage 10 ng 1 × 10 5 3-6 kb ~ 1% 

Pulsed field separated chromosome 50 ng 1 × 10 5 1-2 kb < 1% 

Marine environmental phage 100 ng 1 × 10 6 1-2 kb < 1% 

Cultured Roseophage 100 ng 1 × 10 6 1-2 kb < 1% 

Exiguobacterium 200 ng 5 × 10 5 1-2 kb < 1% 

Streptococcus 200 ng 5 × 10 5 2-4 kb < 1% 


Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 




89 


6


6 

90 Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 


GapFree Shotgun Libraries 

GapFree cloning uses Lucigen's revolutionary pSMART transcription-free vectors (US Pat. 6,709,861) to 

achieve high cloning efficiencies and unprecedented insert stability. As a result, cloning gaps, sequenc- 

ing gaps, and deleted or rearranged sequences are dramatically reduced. We have developed innova- 

tive solutions for cloning a variety of previously "unclonable" DNAs, including: 

• Toxic coding sequences 

• Strong promoters 

• AT- or GC-rich DNA 

• Modified bases 

• Repeats or hairpins 

• Large fragments (>10 kb) 

• Trace amounts of precious sample 

Lucigen is the exclusive supplier of custom shotgun libraries prepared with the 

CloneSmart® technology. 

What you get: 

• Lucigen will shear, end repair, size select, ligate and transform your DNA sample (see fig. 8) 

• Construction of small or large insert, unbiased, and highly complex genomic library 

• Guarantee of >50,000 recombinants per library, with typical yields of >500,000 recombinants 

pSMART 

Small Insert 

Shotgun 

≤ 15 kb 




Send in your sample 

Lucigen’s GapFree 

DNA Libraries Service 

pJAZZ 

Large Insert 

≤ 30 kb 

Result 

Figure 8. Construction of GapFree DNA libraries. 


pSMART-FOS 

Fosmid 

≤ 40 kb 

pSMART-BAC 

BAC 100-200 kb


cDNA Library Construction 

Lucigen’s full-length-enriched cDNA libraries are manufactured by using a technology that combines 

cDNA synthesis and amplification. This results in representative cDNA enriched with full-length 

sequences even from small amounts of starting material. 

• cDNA libraries from any starting material. 

• Full-length cDNA library created in the vector of your choice. 

• Quality control includes library titer determination and verification that >90% 

of clones contain inserts. 

After cDNA synthesis, the double stranded cDNA is size fractionated, cloned (directionally or 

non-directionally) into Lucigen’s pSMART® cloning vectors or into an appropriate customer-supplied 

plasmid vector, and transformed into E. coli. All libraries are made to customer specifications. 

What you get: 

• You receive a glycerol stock (in one or more aliquots) transformed with the complete cDNA library. 

• We can also supply cDNA libraries arrayed in 96- or 384-well plates and other optional tools. 

• A complete report including results of titer and size verification is also provided. 

Metagenomic BAC and Fosmid Libraries 

Lucigen’s full-length-enriched cDNA libraries are manufactured by using a technology that combines 

cDNA synthesis and amplification. This results in representative cDNA enriched with full-length se- 

quences even from small amounts of starting material. 

• High molecular weight DNA prepared from exotic sources. 

• Large insert libraries produced from minimal material. 

Lucigen has successfully developed proprietary techniques for preparing High Molecular Weight 

(HMW) genomic DNA from many sources including collected microbe samples from plant and animal 

hosts; soil samples; marine species: plankton, algae, sponges; and other environmental samples; and 

extremophiles. 

The high quality HMW genomic DNA can be used to manufacture Random Shear BAC and fosmid 

libraries (Fig. 10). 

What you get: 

• All BAC/fosmid libraries are delivered as frozen clones in 384- or 96-well plates with barcode labels 

on the lids and sides and shipped in Lucigen’s library storage boxes on dry ice. 

• A library construction report, technical support, and the directions for safe handling of the custom 

BAC library are also provided. 


kb 

4 

3 

2 

M 

Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

1 

Randomly selected cDNA 

Figure 9. Example of randomly selected cDNA clones 

constructed in pSMART-LCKan vector digested with 

EcoRI, the insert sizes range 1~3kb. M=500bp DNA 

Ladder. 

M 

Micro-Algea Random Shear BAC Clones Cut with Notl 

M 

kb 

200 

150 

100 

50 

Figure 10. High Molecular Weight genomic DNA was 

isolated from micro-algae, randomly sheared, size-selected 

100~300 kb, and cloned into the pSMART BAC 

vector. DNA from minipreps was digested with NotI to 

excise inserts. The vector band is visible at 7 kb. 




91 


6


6 

4x4 Spotting Pattern 

1 2 1 3 

4 5 

P24 

P24 

6 

6 8 3 

4 7 5 

7 

2 

8 

Field 1 

(Plate #1~8) 

Field 4 

(Plate #25~32) 

A1 

A1 

A1 

9 10 9 

11 

12 13 14 15 

14 16 11 10 

12 15 13 16 

Field 2 

(Plate #9~16) 

Field 5 

(Plate #33~40) 

92 Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

A1 

A1 

17 18 17 19 

20 21 22 23 

22 24 19 18 

20 23 21 24 

Field 3 

(Plate #17~24) 

Field 6 

(Plate #41~48) 

Figure 11. Example of a Lucigen gridded filter 

(4x4). 

The membrane (filter) is divided into 6 fields. The spacing 

of colonies is slightly wider between the fields. Each 

field contains 384 squares. The 384 squares represent 

the row and column identification of the BAC clone. 

Within each square there are 16 positions where 8 

clones are spotted in duplicate. The pattern of the spotted 

clones will generate the plate address of the BAC. 

The numbers in the spotting pattern indicate the plate 

numbers. Each grid corresponds to each well of the 

original 384-well plates, starting from A1 from the lefttop 

corner. Each Lucigen filter includes the identification 

code of every clone within the grid. 

Figure 12. A robot picking pin picks a BAC clone from a 

Q-tray plate of Genetix robot. 

A1 

A1 

LuTLBAC1-48 4x4 Set 1 

LIBRARY SCREENING 

Gridding & High Density Colony Filters 

To facilitate screening large numbers of DNA clones, Lucigen can robotically print viable, bacterial cells 

from 96- or 384-well plates onto high-density nylon filters. 

What you get: 

• Your choice of two different sizes and densities of nylon filters 

• Each clone printed in duplicate on filters prepared for your hybridization 

• Automated process ensures high accuracy and quality of your arrays 

• Each filter can be screened 3-5 times for repeat experiments 

• Easy identification and retrieval of original clones from plates 

Table 3 Typical High Density Colony Filters* 

Filter Type Filter Size # of Fields Pattern Spotting # of Clones # of Spots 

Automated Colony Picking, Re-arraying, & Duplicating 

Lucigen offers custom colony picking, re-arraying and duplicating, either as a stand-alone service or as 

part of our library construction services (Random Shear or conventional BAC, fosmid, GapFree shot- 

gun, and NanoClone® libraries). Our well-trained engineers and technicians achieve the highest robot 

picking accuracy and guarantee a library array without empty wells. 

• Automated colony picking transferred from solid to liquid medium 

in plate format 

• Genetix picking robot assesses roundedness, size and color for 

optimal selection 

What you get: 

• Your directly transformed clones or frozen glycerol stocks are grown on agar trays 

• Robotically picked clones transferred to 96 or 384-well plates for further growth 

• BAC libraries delivered to you as frozen clones in chosen plate size – no empty wells 

• Average BAC insert is >100kb for random shear or for conventional BAC clones 




1 22.2cm×22.2cm 6 4×4 Double 18,432 36,864 

2 22.2cm×22.2cm 6 3×3 Double 9,216 18,432 

3 8cm×12cm 1 4×4 Double 3,072 6,144 

4 8cm×12cm 1 3×3 Double 1,536 3,072 

*Custom spotting pattern options are available. 


LIBRARY SCREENING 

Pooling & Superpooling 

Lucigen’s BAC library pooling and superpooling system enables quick and efficient identification of a 

sequence of interest from even large libraries. 

What you get: 

• Pooled clone DNA from your library in a grid of 96-well plates 

• Samples organized to allow the library address of your sequence in as little as 59 PCR reactions 

• Most clones can be indentified in only two rounds 

• Custom pooling patterns designed by customer are available 

Custom Library Screening 

Lucigen provides a library screening service to assist clients with identifying BAC and/or fosmid clones 

from custom BAC/fosmid library constructed at Lucigen or other library supplied by the client. 

• Library screening via oligonucleotide (overgo) probes hybridized to customprepared 

nylon filters 

• 18,432 BAC/fosmid clones spotted in duplicate on each 22 x 22 cm filter 

• Positive clones identified via 32P radioactive probe hybridization 

• Available as a standalone option or in combination with Lucigen’s other services 

What you get: 

• Receive your positive clone(s) in glycerol stock 

• A complete report of screening results is provided 


Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 




93 


6


6 

94 Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

DNA PREP 

High Molecular Weight genomic DNA preparation 

Ultra pure High Molecular Weight genomic DNA 

• Genomic DNA up to Megabase-size provided from any source. 

• DNA quality suitable for any application. 

Proprietary techniques result in High Molecular Weight genomic DNA from: 

Microbes Plants Animals 

Any species Any tissue or cell BAC clones 

DNA is prepared in solution or agarose plugs (optional) to ultra-high quality standards and is ready for 

any research use including: 

• Clone-free library construction for Next-Gen sequencing 

• Large-insert cloning in pSMART® BAC or pJAZZ® Vectors. 

• Other cloning, PCR, etc. 

What you get: 

• Ultrapure genomic DNA up to Megabase-size ready for your next application 

• Samples provided in TE buffer 

• Complete report of quality testing is provided with each sample 

HMW genomic DNA 

from low-melting agarose. 

kb 

500 

300 

100 

� 




Figure 13. Example of HMW genomic DNA preparation. 

Liquid HMW genomic DNA a�er 

electroelution and dialysis. 

Left Panel: HMW genomic DNA in Low-Melting-Temperature agarose 

plugs, which is >1Mb, up to entire chromosome/genome size. 

Right Panel: Liquid HMW genomic DNA with up to 500 Kb-size. 

� 


kb 

16 

10 

2

DNA PREP 

BAC DNA Preparation 

Ultra-high quality BAC DNA prepared in the high quantities needed for: 

• Subcloning, constructing clone-free libraries for Next-gen sequencing, etc. 

from individual BAC clones 

• BAC DNA pooling–can be customized to fit your needs 

• Sanger BAC end sequencing, BAC End sequencing.* 

*With Lucigen’s CopyRight BAC cloning system, sufficient DNA for forward and reverse reactions with 

up to 95% success and reads >800bp can be produced. 

What you get: 

• Large DNA fragments produced from BAC DNA or BAC clones. 

• Controlled quantity and quality of DNA with optimized digestion conditions, 

followed by CHEFF gel electrophoresis. 

• Gel-purified fragment is provided in TE or microinjection buffer (10mM Tris- 

HCl, pH 7.5, 0.1mM EDTA, 100mM NaCl). 


kb 

300 

200 

100 

M1 

BAC 

Figure 14. Example of purification of a 160 kb BAC fragment. 

BAC. Left Panel: BAC DNA digested with NotI, 

M1=Lambde ladder DNA marker, v=vector band ~7 kb. 

Right Panel:=LF: purified ~160-kb Large Fragment BAC, 

M2=supercoil DNA marker. Arrows indicate the large 

fragment BAC before and after purification. 

Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

v 

LF 

M2 

16 

10 

2 




95 


6

Appendix 

Product Index by 

Product Name ..................................98 

Product Index by 

Catalog Number ..............................99 

Lucigen Publications .....................100 

Warranty and Disclaimers ............101 

Reference .......................................102 


Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

Solutions 




97

PRODUCT INDEX 

By Product Name 

BAC-Optimized Replicator v2.0 Cells . . . . .47 

BigEasy® Long PCR Cloning Kits . . . . . . . . . . . .32 

BigEasy TSA Electrocompetent Cells ..32, 34 

BigEasy v2.0 Linear Cloning System ........34 

Bst Adapter Fill-In Kit .....................37 

Bst DNA Polymerase, Exo Minus. . . . . . . . . . . .19 

CAZyme Carbohydrases . . . . . . . . . . . . . 76-77 

CloneDirect Rapid Ligation Kit ...........36 

CloneSmart® Blunt Cloning Kits ............28 

CopyRight® Cloning Kits. . . . . . . . . . . . . . . . . . .33 

Competent Cells for Cloning ..............40 

Competent Cell Service . . . . . . . . . . . . . . . . . . .44 

Custom Services ..........................80 

DNA Polymerase I Large (Klenow) 

Fragment, Exonuclease Minus ..............70 

DNATerminator® End Repair Kit ......... 35+ 

E. cloni 10G BAC-Optimized 

Electrocompetent Cells ...................47 

E. cloni® 10G & 10GF´ Competent Cells ....42 

E. cloni® 5-alpha Competent Cells ..........44 

E. cloni® EXPRESS Competent Cells . . . . . . . . .61 

EconoTaq® DNA Polymerase . . . . . . . . . . . . . . .14 

EconoTaq PLUS 2X Master Mix . . . . . . . . . . . . .13 

EconoTaq PLUS GREEN 2X Master Mix ......13 

Endura Competent Cells . . . . . . . . . . . . . . . . .46 

ER2738 Electrocompetent Cells. . . . . . . . . . . .45 

Exonuclease I ............................73 

Exonuclease III. . . . . . . . . . . . . . . . . . . . . . . . . . . .74 

98 Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

Expressioneering Technology ............50 

Expresso® Cloning & Expression Systems . . . .51 

Expresso® T7 Cloning & Expression Systems 51 

Expresso® Rhamnose 

Cloning & Expression System ..............54 

Expresso® CMV 

Cloning & Expression System ..............56 

Expresso® SUMO Solubility Tag ............52 

Gel-Ready DNA Ladders. . . . . . . . . . . . . . . . .37 

GC Cloning & Amplification Kits ...........31 

Lambda Exonuclease .....................73 

Library Construction Service. . . . . . . . . . . . . . .81 

M-MuLV Reverse Transcriptase ............68 

NextGen Sequencing Services .............85 

NxGen Genomic Grade Enzymes ..........66 

OverExpress Competent Cells. . . . . . . . . . . .59 

PCR Grade dNTPs . . . . . . . . . . . . . . . . . . . . . . . .20 

Phage Display Electrocompetent Cells ......45 

PCRTerminator® End Repair Kit ............35 

pEZ BAC Vector. . . . . . . . . . . . . . . . . . . . . . . . .33 

pEZSeq Blue/White Blunt Cloning Kits ....29 

phi29 DNA Polymerase ...................68 

pInvRecA Vector . . . . . . . . . . . . . . . . . . . see web 

pJAZZ® GC Vector . . . . . . . . . . . . . . . . . . . . . . . .32 

pJAZZ® Vectors. . . . . . . . . . . . . . . . . . . . . . . .32,34 

pJW168 Vector. . . . . . . . . . . . . . . . . . . . . see web 

pLEXX-AK Vector .................see web 

pRANGER-BTB Vectors .............see web 





pSMART® BAC Vector ....................33 

pSMART GC Vector ......................30 

pSMART Vectors .........................27 

Pyrophage® 3173 DNA Polymerase 

(Wild Type) ..............................15 

Pyrophage® 3173 DNA Polymerase (Exo-) ..18 

PyroPhosphatase . . . . . . . . . . . . . . . . . . . . . . . . .75 

PyroScript® RT-PCR 2x Master Mix Kit ......17 

Random Shear BAC Library 

Construction Service ......................81 

RNase I ..................................74 

RNAse Inhibitor ..........................75 

Sequencing Services ......................87 

SS320 Electrocompetent Cells .............45 

T4 DNA Ligase ...........................72 

T4 DNA Polymerase ......................71 

T4 DNA Polymerase, Exonuclease Minus. . . .71 

T4 Polynucleotide Kinase .................75 

T4 RNA Ligase . . . . . . . . . . . . . . . . . . . . . . . . . . .72 

T7 RNA Polymerase ......................69 

T7 DNA Polymerase ......................69 

Taq98 Hot Start . . . . . . . . . . . . . . . . . . . . . . . . .12 

TaqSelect DNA Polymerase ..............16 

Terminal Transferase .....................70 

TG1 Electrocompetent Cells . . . . . . . . . . . . . . .45 

UltraClone DNA Ligation Kit .............36

PRODUCT INDEX 

By Catalog Number 

30027 ...............19 

30028 ...............19 

30029 ...............20 

30030 ...............20 

30031 ...............14 

30032 ...............14 

30033 ...............13 

30035 ...............13 

30041 ...............16 

30051 ...............15 

30053 ...............18 

30055 ...............17 

30057 ...............12 

30061 ...............75 

30072 ...............18 

30074 ...............75 

30081 ...............71 

30085 ...............71 

30095 ...............70 

30104 ...............74 

30221 ...............68 

30222 ...............68 

30223 ...............69 

30224 ...............69 

30225 ...............70 

30241 ...............72 

30243 ...............72 

30244 ...............72 

30261 ...............73 

30262 ...............73 

30263 ...............74 

30281 ...............75 

30501 ...............77 

30502 ...............77 

30533 ...............77 

30534 ...............77 

30535 ...............77 

30553 ...............77 

30554 ...............77 

30555 ...............77 

30556 ...............77 

30557 ...............77 

30558 ...............77 

30559 ...............77 

30560 ...............77 

30561 ...............77 

30572 ...............77 

30610 ...............77 

30620 ...............77 

30630 ...............77 

30640 ...............77 

30650 ...............77 

30801 ...............55 

30805 ...............55 

31511 ...............77 

31531 ...............77 

31532 ...............77 

31551 ...............77 

31552 ...............77 

31571 ...............77 

31591 ...............77 

31592 ...............77 

40002 ...............36 

40003 ...............36 

40004 ...............36 

40008 ...............36 

40012 ...............36 

40020 ...............36 

40031 ...............37 

40035 ...............35 

40037 ...............35 

40041 ...............28 

40052 ...............28 

40054 ...............28 

40063 ...............28 

40065 ...............28 

40074 ...............28 

40076 ...............28 

40300 ...............28 

40311 ...............28 


40313 ...............28 

40322 ...............28 

40324 ...............28 

40333 ...............28 

40335 ...............28 

40464 ...............29 

40475 ...............29 

40486 ...............29 

40488 ...............29 

40494 ...............29 

40496 ...............29 

40500 ...............29 

40501 ...............29 

40512 ...............29 

40514 ...............29 

40524 ...............29 

40526 ...............29 

40704 ...............28 

40706 ...............28 

40708 ...............28 

40717 ...............28 

40719 ...............28 

40728 ...............28 

40730 ...............28 

40731 ...............31 

40732 ...............31 

40733 ...............31 

40736 ...............31 

40737 ...............31 

40738 ...............31 

40741 ...............31 

40742 ...............31 

40743 ...............31 

40821 ...............28 

40832 ...............28 

40834 ...............28 

40843 ...............28 

40845 ...............28 

40854 ...............28 

40856 ...............28 

42007 ...............33 

42009 ...............33 

42012 ...............33 

42016 ...............33 

42027 ...............33 

42030 ...............33 

42031 ...............33 

42032 ...............33 

42102 ..........see web 

42104 ..........see web 

42106 ..........see web 

42108 ..........see web 

42200 ..........see web 

42205 ..........see web 

43018 ...............34 

43024 ...............34 

43036 ...............34 

43042 ...............34 

43054 ...............32 

43066 ...............32 

49000 ...............55 

49001 ...............55 

49002 ...............55 

49003 ...............55 

49010 ...............55 

49011 ...............55 

49012 ...............55 

49013 ...............55 

49021 ...............55 

49022 ...............55 

49031 ...............57 

50010 ...............37 

50020 ...............37 

60051 ...............43 

60052 ...............43 

60061 ...............43 

60062 ...............43 

60080 ...............43 

60081 ...............43 

60096 ...............43 

Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

60106 ...............43 

60107 ...............55 

60108 ...............43 

60110 ...............43 

60117 ...............43 

60210 ............33,47 

60215 ...............47 

60224 ............32, 34 

60240 ...............46 

60241 ...............46 

60242 ...............46 

60300 ...............62 

60311 ...............62 

60324 ...............62 

60333 ...............62 

60341 ...............60 

60343 ...............60 

60345 ...............60 

60347 ...............60 

60350 ...............60 

60401 ...............62 

60413 ...............62 

60425 ...............62 

60430 ...............62 

60435 ...............55 

60442 ...............60 

60444 ...............60 

60446 ...............60 

60448 ...............60 

60452 ...............60 

60500 ...............45 

60502 ...............45 

60512 ...............45 

60522 ...............45 

60602 ............43, 44 

80026 ............43, 45 

80030 ...............60 




99

SELECTED LUCIGEN PUBLICATIONS 

Breitbart, M., Salamon, P., Andresen, B., Mahaffy, J.M., Segall, A.M., Mead, D., Azam, F., and Rohwer, F. (2002). Genomic analysis of uncultured marine viral 

communities. Proc. Nat. Acad. Sci. 99: 14250-14255. 

Breitbart, M., Wegley, L., Leeds, S., Schoenfeld, T., Rohwer, F. (2004). Phage community dynamics in hot springs. Appl Environ Microbiol. 70:1633. 

Brumm P, Hermanson S, Hochstein B, Boyum J, Hermersmann N, Gowda K, Mead D. (2011) Mining Dictyoglomus turgidum for enzymatically active carbohydrases. 

Appl Biochem Biotechnol. 163:205-214. 

Brumm P, Mead D, Boyum J, Drinkwater C, Gowda K, Stevenson D, Weimer P. (2011) Functional Annotation of Fibrobacter succinogenes S85 Carbohydrate 

Active Enzymes. Appl Biochem Biotechnol. 163:649-57 

Carrias A, Welch TJ, Waldbieser GC, Mead DA, Terhune JS, and Liles MR (2011) Comparative Genomic Analysis of Bacteriophages specific to the Channel 

Catfish Pathogen Edwardsiella ictaluri. Virology J. In Press. 

Chang Y, Mead DA, Dhodda V, Brumm P, and Fox BG. (2009) One-plasmid tunable co-expression for mycobacterial protein-protein interaction studies. 

Protein Sci. 18:2316-2325. 

Dhodda V, Godiska R, Vanwye JD, Mead D, Hochstein R, Sheets L, Zande SV, Niebauer C, Crawford DL, Oleksiak MF. (2010) ExCyto PCR Amplification. PLoS 

One 8:5(9). 

Gao D, Uppugundla N, Chundawat SP, Yu X, Hermanson S, Gowda K, Brumm P, Mead D, Balan V, Dale BE. (2011) Hemicellulases and auxiliary enzymes for 

improved conversion of lignocellulosic biomass to monosaccharides. Biotechnol Biofuels. 4:5. 

Godiska R, Mead DA, Dhodda V, Hochstein R, Karsi A, Usdin K, Entezam A, Ravin N. (2010) Linear plasmid for cloning large or repetitive sequences in E. coli. 

Nuc. Acids Res. 38:e88. 

Godiska, R., Mead, D., Dhodda, V., Hochstein, R., Karsi, A., Usdin, K., Ravin, N. (2008). "Linear vector for cloning large or repetitive DNAs in E. coli." In DNA III: 

Dealing with Difficult Templates. ( J. Kieleczawa, ed.), Jones and Bartlett Publishers, Sudbury, MA. 

Godiska, R., Patterson, M., Schoenfeld, T., Mead, D.A. (2005). “Beyond pUC: Vectors for Cloning Unstable DNA.” In DNA Sequencing: Optimizing the Process 

and Analysis. ( J. Kieleczawa, ed.), Jones and Bartlett Publishers, Sudbury, MA. 

Heidelberg, J.F., Nelson, W.C., Schoenfeld, T., Bhaya, D., (2009). Germ warfare in a microbial mat community: CRISPRs provide insights into the co-evolution of 

host and viral genomes. PLoS ONE. 4(1):e4169. 

Kakirde KS, Wild J, Godiska R, Mead DA, Wiggins AG, Goodman RM, Szybalski W, and Liles MR (2010) Gram negative shuttle BAC vector for heterologous 

expression of metagenomic libraries. Gene. 2010 Nov 25. [Epub ahead of print] PubMed PMID:21112378. 

Pride, DT., Schoenfeld, T. (2008). Genome signature analysis of thermal virus metagenomes reveals Archaea and thermophilic signatures. BMC Genomics. 

9:420. 

Reysenbach AL, Hamamura N, Podar M, Griffiths E, Ferreira S, Hochstein R, Heidelberg J, Johnson J, Mead D, Pohorille A, Sarmiento M, Schweighofer K, 

Seshadri R, Voytek MA. (2009) Complete and draft genome sequences of six members of the Aquificales. J Bact. 191:1992-1993. 

Samuels, M., Leamon, J., Rothberg, J., Godiska, G., Schoenfeld, T., Mead, D. (2009). "New Paradigms in Droplet Based Microfluidics and DNA Amplification." In 

Automation in Genomics and Proteomics: An Engineering Case-Based Approach (G Alterovitz, M Ramoni, R Beson ed.) Wiley. 

Schoenfeld T, Liles M, Wommack EK, Polson SW, Godiska R, and Mead D. (2010) Functional viral metagenomics and the next generation of molecular tools. 

Trends in Microbiology. 18:20-29. 

Schoenfeld, T., Patterson, M., Richardson, P.M., Wommack, K.E., Young, M., Mead, D. (2008). Assembly of viral metagenomes from yellowstone hot springs. 

Appl Environ Microbiol. 74:4164. 

Srinivasiah, S., Bhavsar, J., Thapar, K., Liles, M., Schoenfeld, T., Wommack, K.E. (2008). Phages across the biosphere: contrasts of viruses in soil and aquatic 

environments. Res Microbiol. 159:349. 

Suen G, Weimer PJ, Stevenson DM, Aylward FO, Boyum J, Deneke J, Drinkwater C, Ivanova NN, Mikhailova N, Chertkov O, Goodwin LA, Currie CR, Mead D, 

and Brumm PJ. 2011. The Complete Genome Sequence of Fibrobacter succinogenes S85 Reveals a Cellulolytic and Metabolic Specialist. PLoS ONE, 

In press. 

Wommack, K.E., Bench, S.R., Bhavsar, J., Mead, D., Hanson, T. (2009). "Isolation independent methods of characterizing phage communities: Characterizing a 

metagenome." In Bacteriophages: Methods and Protocols (A. Kropinski, M. Cloike eds.) Humana Press. 

100 Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 





WARRANTY AND DISCLAIMERS 

Patents and Limited Label License 

Lucigen’s vectors incorporating the CloneSmart® transcription-free cloning technology 

are the subject of U.S. Patent No. 6,709,861 and pending patent applications 

owned by Lucigen Corporation. The consideration paid for these products 

grants a Limited License to use the purchased amount of products pursuant to 

the terms set forth in the Limited Label License. Except where stated otherwise, 

the Limited License specifically excludes manufacture of additional vector 

derived from these kits. Academic, Not-for-Profit and For-Profit institutions acquire 

certain limited nontransferable rights with the purchase of these products. 

A complete description of the Limited License rights and limitations accompanies 

these products and is available from Lucigen on request. Broader licenses to use 

these patented vectors for commercial purposes are also available. Please contact 

Lucigen for details. 

Lucigen’s CopyRight® vectors and Replicator competent cells are covered by 

U.S. Patent No. 5,874,259. Lucigen's GC Cloning Kits & Vectors are covered by 

U.S. Patent No. 7,723,103. By purchasing these products, the purchaser receives a 

limited use license for life science research applications. 

OverExpress, HI-Control BL21(DE3), and E. cloni® EXPRESS products are sold 

under a license issued to Lucigen Corporation by Brookhaven Science Associates, 

LLC (BSA) for noncommercial research purposes only. A separate license is 

required for any commercial use, including the use of these materials for research 

purposes or production purposes by any commercial entity. Information about 

commercial licenses may be obtained from: 

Office of Technology Commercialization and Partnership 

Brookhaven National Laboratory, Bldg. 490-C, P. O. Box 5000 

Upton, New York 11973-5000 

Telephone (631)-344-7134 

In using this product, you agree that OverExpress, HI-Control BL21(DE3) and, 

E. cloni® EXPRESS cells or its derivatives containing the cloned gene for T7 RNA 

Polymerase may not be distributed further to third parties outside of your laboratory, 

unless the recipient receives a copy of this limited label license and agrees 

to be bound by its terms. 

OverExpress products are sold for research use only by nonprofit and commercial 

entities under an exclusive license issued to Lucigen Corporation by Imaxio, 

S.A. under U.S. Patent No. 6,361,966, AU Patent No. 716694, and pending 

patent applications. Commercial use of OverExpress to produce a product or 

service for sale requires a separate license. Contact: 

IMAXIO Business Development Director 

Biopôle Clermont-Limagne 

63360 Saint Beauzire, France 

Fax: + 33 473 644 393 

E-mail: overexpress@imaxio.com 

The 6xHis tag is licensed from Hoffmann-La Roche, Inc., Nutley, NJ and/or 

Hoffmann-LaRoche Ltd., Basel, Switzerland and is provided only for the use in 

research. Information about licenses for commercial use is available from 

QIAGEN GmbH 

QIAGEN Strasse 1 

D-40724 Hilden, Germany 

SUMO Protease is manufactured and supplied by LifeSensors, Inc. 

Some applications in which Lucigen’s EconoTaq® DNA Polymerase can be used 

may be covered by patents issued and applicable in the United States and certain 

other countries. Because purchase of this product does not include a license 

to perform any patented application, users of these products may be required to 

obtain a patent license depending upon the particular application in which the 

product is used. The PCR process is the subject of European Patent Nos. 201,184 

and 200,262 owned by Hoffman-LaRoche. Those patents expired on March 28, 

2006. The corresponding PCR process patents in the United States expired on 

March 29, 2005. 


Trademarks 

Lucigen, BigEasy, ClonePlex, CloneSmart, CopyRight, DNATerminator, E. cloni, 

EconoTaq, Expresso, Lucigen, NanoClone, PCR Terminator, pJAZZ, pSMART, 

PyroPhage, pETite, and PyroScript are registered trademarks of Lucigen Corporation. 

bSMART, Blue/White cloning without the blues, ChimeraFree, CloneDirect, 

cSMART, eLucidations, eLucidator, Endura, GapFree, HI-Control, NxGen, 

PCR SMART, pEZ, pEZSeq, pGC, pLEXX-AK, pRANGER, pRham, Replicator, 

Taq98, The Cloning Company, The Molecular Cloning Company, Transformance, 

TSA, and UltraClone are trademarks of Lucigen Corporation. Hydroshear and 

GeneMachines are trademarks of Genomic Solutions, Inc. Vent is a trademark of 

New England Biolabs, Inc. pGEM is a trademark of Promega Corporation. TOPO, 

TA, Zero Blunt, DH5a, and DH10B are trademarks of Invitrogen Corporation. 

pCC1BAC is a trademark of Epicentre Technologies Corporation. MicroPulser, 

and Gene Pulser are trademarks of BioRad Laboratories, Inc. OverExpress is a 

trademark of Imaxio, S.A. 

Use Restriction 

Lucigen’s products are for research or laboratory use only and are not to be administered 

to humans or animals or used for pharmaceutical, in vitro diagnostic, 

or commercial purposes without written permission. Inquiries related to additional 

uses of Lucigen’s products are welcomed. Lucigen shall not be responsible 

for injury or damages resulting from the use or misuse of any of its products. 

Persons using the products should be technically qualified as defined in 40 C.F.R. 

§ 720.3(ee) and should follow the Guidelines for Research involving Recombinant 

DNA Molecules (N.I.H. Guidelines) as presented in the Federal Register, July 

5, 1994 (59 FR 34496) and any amendments thereto. 

Warranties 

Lucigen warrants that its products will conform to the standards stated in its 

product specification sheets in effect at the time of shipment. Lucigen will 

replace, free of charge, any product that does not conform to the specifications. 

This warranty limits Lucigen’s liability only to the replacement of the product. 

THIS WARRANTY IS EXCLUSIVE, AND LUCIGEN MAKES NO OTHER WAR- 

RANTY, EXPRESS OR IMPLIED, INCLUDING WITHOUT LIMITATION, ANY IM- 

PLIED WARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR 

PURPOSE. The stated express warranties, and the remedy provided for breach 

thereof, are in lieu of all other liability or obligations of Lucigen for any damages 

whatsoever arising out of or in connection with the delivery, use, performance, 

or the inability to use any of its products. IN NO EVENT SHALL LUCIGEN BE 

LIABLE UNDER ANY LEGAL THEORY (INCLUDING BUT NOT LIMITED TO 

CONTRACT, NEGLIGENCE, STRICT LIABILITY IN TORT, OR WARRANTY OF 

ANY KIND) FOR ANY INDIRECT, SPECIAL, INCIDENTAL, CONSEQUENTIAL, 

OR EXEMPLARY DAMAGES (INCLUDING BUT NOT LIMITED TO LOST PROF- 

ITS). Without limiting the effect of the preceding sentence, Lucigen’s maximum 

liability, if any, shall not exceed the purchase price paid by buyer for the product. 

Exclusive Terms of Sale 

Lucigen does not agree to and is not bound by any other terms or conditions, 

unless those terms or conditions have been expressly agreed to in writing by a 

duly authorized officer of Lucigen. 

Prices are subject to change without notice. 

All material in this catalog is ©2012 Lucigen Corporation, 

Middleton, Wisconsin USA. 

Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 




101

Avoid after initiating Met 

Val 

Arg 

Best Codons in E. coli 

AA Codon AA Codon 

Ala GCG 

Gly GGC 

Reference 

Increase your chance of success 

GGU 

Lys 

Phe 

Leu 

Trp 

102 Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

Tyr 

Ser AGC 

Pro CCG 

Leu CUG 

Thr ACC 

Tip! 




Genetic Code 

Valine 

Arginine 

Biochemical characteristics of Amino Acids 

Aliphatic 

Aromatic 

Serine 

Alanine 

Lysine 

M 

A 

C 

U 

G 

A 

C 

G 

U 

G 

Asparagine 

Aspartic 

Acid 

U 

A 

I 

A G 

C 

U 

G 

A 

C 

U 

L 

CS-S 

F 

Tiny 

V 

A G 

Hydrophobic 

C 

C 

A 

U 

G 

G 

�reonine 

Glutamic 

Acid 

A 

C 

U 

G U 

A C 


C 

U 

www.lucigen.com 

Methionine 

G 

Glycine 

U 

A 

Small 

P 

A 

G 

S N 

Y 

C 

Isoleucine 

C 

W 

A G 

U 

T 

Phenylalanine 

C 

A 

G 

U U 

C C 

A 

G 

U 

C 

G A 

G 

A 

CS-H 

H 

C 

Arginine 

Leucine 

U C A G 

U 

U 

K 

G 

A 

R 

C 

Glutamine 

Serine 

U 

G 

D 

A 

C 

Histidine 

A 

G 

U 

E 

Tyrosine 

U 

G 

C 

A 

A 

C 

Stop 

G 

U 

Proline 

Cysteine 

Q 

Stop 

Tryptophan 

Leucine 

+ Positive 

Polar 

- Negative

Genome Copy Numbers 

Organism 

www.lucigen.com 

haploid 

genome 

size (bp) 

Average weight of DNA 

Average weight of a DNA basepair 

(sodium salt) = 650 daltons 

1.0 A 260 unit ds DNA = 50 µg/ml = 

0.15 mM (in nucleotides) 

1.0 A 260 unit ss DNA = 33 µg/ml = 


1.0 A 260 unit ss RNA = 40 µg/ml = 


"C-value 

g/copy" copy/ng copy/pg 

Homo sapiens 3.00E+09 3.3E-12 310 0.3 

Zea mays 2.30E+09 2.5E-12 400 0.04 

D. melanogaster 1.80E+08 2.0E-13 5100 5.1 

S. cerevisiae 1.22E+07 1.3E-14 76000 76 

S. pombe 1.41E+07 1.5E-14 65000 65 

E. coli K-12 4.60E+06 5.0E-15 200000 200 

Herpes Virus 1.53E+05 1.7E-16 6.0E+06 6,000 

Lambda phage 4.85E+04 5.3E-17 1.9E+07 19,000 

pUC19 2.30E+03 2.5E-18 4.0E+08 400,000 

MW of a double-stranded DNA 

molecule = 

(# of base pairs) × (650 daltons/ 

base pair) 

Moles of ends of a double-stranded 

DNA molecule = 

2 × (grams of DNA) / 

(MW in daltons) 


1 µg of 1000 bp DNA = 1.52 pmol = 

9.1 × 10 11 molecules 

1 µg of pUC18/19 DNA (2686 bp) = 

0.57 pmol = 3.4 × 10 11 molecules 

1 µg of pBR322 DNA (4361 bp) = 

0.35 pmol = 2.1 × 10 11 molecules 

1 µg of M13mp18/19 DNA (7249 

bp) = 0.21 pmol = 1.3 × 10 11 

molecules 

1 µg of l DNA (48502 bp) = 0.03 

pmol = 1.8 × 10 10 molecules 

Orders: Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

Reference 

1 pmol of 1000 bp DNA = 

0.66 µg 

1 pmol of pUC18/19 DNA 

(2686 bp) = 1.77 µg 

1 pmol of pBR322 DNA 

(4361 bp) = 2.88 µg 

1 pmol of M13mp18/19 DNA (7249 

bp) = 4.78 µg 

1 pmol of l DNA (48502 bp) 

= 32.01 µg 

1.0 kb DNA = coding capacity for 

333 amino acids 

~37,000 dalton protein 

10,000 dalton protein ~270 bp 

DNA 

Online tools to make your work easier. 

50,000 dalton protein 

~1.35 kb DNA 

Cloning: lucigen.com/cloningguide 

Go� 

Competent Cells: lucigen.com/compcellguide 




103 

Go�

1: PCR & Amplification 9 

2: Cloning Kits & Vectors 23 

3: Competent Cells 39 

4: Protein Expression 49 

5: Enzymes 65 

6: Library Services 79 

appendix 97 

Orders: 

Online: www.lucigen.com 

lucigen@lucigen.com 

Phone: 608 831 9011 

888 575 9695 

FAX: 608 831 9012 

Tech Support: 

Phone: 608 831 9011 

E-mail: techserv@lucigen.com 

www.lucigen.com

Lucigen 2012 Catalog Update

Create successful ePaper yourself

Delete template?

Save as template?