EPICENTRE Forum 14-2

Volume 14-2 

Featuring: 

New Systems for RNA 

and cDNA Studies 

Introducing the ExactStart 

Full-Length cDNA Library 

Cloning Kit for Obtaining 

Full-Length cDNAs and 

Accurate Mapping of 

Transcript Ends 

Achieve Fast Real-Time PCR 

Results With the New PCR 

MasterMix Kit 

Extract PCR-Ready Plant Genomic 

DNA Using a Simple 8-Minute, 

1-Tube Protocol 

Detect More Genes With the New 

TargetAmp Kit Designed for the 

Illumina ® System 

Cover Photo: La Jolla, CA 

Photograph by: Dilara Begum, 

EPICENTRE Biotechnologies, Madison, WI

Be certain that ZERO 

DNA remains 

NEW! EPICENTRE Biotechnologies’ 

Baseline-ZERO DNase digests doubleand 

single-stranded DNA more completely 

than any other product on the market. 

qPCR Demonstrating DNA Contaminants 

• Removes genomic DNA from RNA prior to 

RT-PCR. 

• Prepares target RNA for cDNA in microarray 

or expression analysis. 

• Eliminates the DNA template following 

in vitro RNA synthesis with T7, T3, or SP6 

Phage RNA Polymerases. 

• Removes ssDNA and dsDNA from viral 

RNA. 

• Eliminates genomic DNA from RNA for 

microinjection and transfection experiments. 

C T 

values from real-time PCR of HeLa RNA preparations treated with various 

DNases. The lower the C T 

value, the greater the amount of residual DNA not 

digested by the indicated DNase. Thus, Baseline-ZERO DNase removed all 

detectable DNA from the RNA sample. The TaqMan probe assay amplifi ed a 

268 bp fragment of beta actin. Samples were run in duplicate. 

To order yours, go to www.EpiBio.com 

and enter code: BZAX3 

• Simple 10 minute heat inactivation.

EPICENTRE® Biotechnologies Forum 

IN THIS ISSUE 

ExactStart Full-Length cDNA Library Cloning Kit: A Rapid and Efficient 

Method to Synthesize Full-Length cDNA for Cloning and Accurate 

Mapping of Transcription Initiation and Polyadenylation Sites. . . . . . . . . 4 

The new ExactStart Full-Length cDNA Library Cloning Kit produces full-length cDNA libraries 

from a variety of eukaryotic sources. These libraries can be used to study alternate 5′ transcriptional 

start and 3′ polyadenylation sites. 

Fast Real-Time PCR with a Standard qPCR Thermocycler! . . . . . . . . . . . 6 

The TAQXpedite GREEN Real-Time PCR MasterMix Kit enables fast real-time PCR (fast 

qPCR) reactions to be performed using standard qPCR instrumentation. 

HOW TO CONTACT US 

Toll-free in the USA: 800-284-8474 

Tel: 608-258-3080 Fax: 608-258-3088 

Mail: 726 Post Rd., Madison, WI 53713 

Website: www.EpiBio.com 

E-mail: forum@EpiBio.com 

Outside the USA: contact your local 

EPICENTRE Distributor. 

www.EpiBio.com/distributors.asp 

EPICENTRE Biotechnologies FORUM 

Editor: Simon T. M. Allard, Ph.D. 

Graphic Designer: Julie Capadona 

Additional Illustrations: Ron Meis 

COMPLIMENTARY 

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©2007 EPICENTRE ® Biotechnologies All rights 

reserved. Publication date: October 2007. Printed in USA. 

EPICENTRE, Ampligase, DuraScribe, and DuraScript 

are registered trademarks and 2-Way, A-Plus, AmpliCap, 

AmpliCap-Max, AmpliScribe, AmpliTherm, APex, 

A-Plus, ArrayPure, BAC-Tracker, BACMAX, BACMAX96, 

BuccalAmp, Catch-All, CircLigase, Colony Fast-Screen, 

CopyControl, CopyCutter, EC100, EC100D, EPI300, 

EPI400, EasyLyse, End-It, EpiBlue, EpiFOS, Epi-Grids, 

EpiLyse, ExactStart, ExtractMaster, EZ::TN, EZ-Tn5, 

FailSafe, Fast-Link, Fast-Screen, FosmidMAX, GELase, 

HK, Hybridase, HyperMu, IsoTherm, Kool, Long-Read, 

MasterAmp, MasterPure, MaxPlax, MessageBOOSTER, 

MessageMAX, MessageMuter, MiniV, MonsterScript, 

mRNA-ONLY, mScript, Mu-End, NC-45, NTPhos, 

Nano-g, OmniCleave, pCC1, pCC1BAC, pCC1FOS, 

pCC2FOS, PCRLyse, PeriPreps, PlasmidMAX, 

Plasmid-Safe, pEpiFOS, pMOD, pPDM, pWEB, 

pWEB-TNC, QuickExtract, R&DNA, Ready-Lyse, 

ReadyPreps, RepliPHI, Restricti-Lyse, RetroAmp, RiboScribe, 

RiboShredder, ScriptCap, ScriptGuard, SequiTherm, 

SequiTherm EXCEL, SoilMaster, T3-Flash, T7-Flash, 

Tagetin, TAQurate, TAQXpedite, TargetAmp, Terminator, 

TransforMax, Transposome, Transposomics, TypeOne, and 

WaterMaster are trademarks of EPICENTRE. RNeasy 

is a registered trademark of Qiagen Corp. GeneChip is a 

registered trademark of Affymetrix. CodeLink and Cy are 

trademarks of GE Healthcare. SuperScript is a registered 

trademark of Invitrogen. SYBR is a registered trademark of 

Molecular Probes, Invitrogen Detection Technologies. Product 

prices and specifications are subject to change without notice. 

Easy and Rapid Extraction of Plant DNA Using the QuickExtract Plant 

DNA Extraction Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 

The QuickExtract Plant DNA Extraction Solution represents a rapid and efficient way to extract 

PCR-ready plant genomic DNA from most plant samples. 

Product Data Sheets for EPICENTRE Biotechnologies’ Products ........ 9 

TargetAmp Nano-g Biotin-aRNA Labeling Kit for the Illumina® System 

TargetAmp 2-Round Biotin-aRNA Amplification Kit 3.0 

EZ-Tn5 Insertion Kit 

EZ-Tn5 In-Frame Linker Insertion Kit 

EZ-Tn5 Promoter Insertion Kit 

EZ-Tn5 Transposon Construction Vectors 

QuickExtract Plant DNA Extraction Solution 

QuickExtract DNA Extraction Solution 1.0 

TAQXpedite PCR System (FAST End-Point) 

TAQXpedite GREEN Real-Time PCR MasterMix Kit 

Ask Frank, by Fred Hyde and Hank Daum . . . . . . . . . . . . . . . . . . . . . . . 17 

Questions about Capping of RNA and in vitro transcription, answered by EPICENTRE’s Technical 

Consultants. 

A Simple Enzymatic Process for Generating mRNA-Enriched Poly(A)- 

Tailed RNA from Total RNA Samples ............................ 18 

EPICENTRE’s mRNA-ONLY Prokaryotic mRNA Isolation Kit with Poly(A)-Tailing provides 

a simple enzymatic process for generating mRNA-enriched poly(A)-tailed RNA from total RNA 

samples. 

Protein Expression from a Chromosomally-Inserted Transposon. . . . . . 20 

Chromosomal protein expression using a transposon-mediated insertion is an alternative for highlevel 

expression of proteins in E. coli. Antibiotics or other selective pressures are not required to 

maintain the transposon insertion. 

Isolate Intron Lariat Loops with RNase R. ........................ 22 

Ribonuclease R (RNase R) from E. coli, is a magnesium-dependent 3′→5′ exoribonuclease that 

digests essentially all linear RNAs but does not digest lariat or circular RNA structures. 

Talk tidbits ................................................ 23 

Featuring— 

TransforMax EC100 E. coli competent cells 

Fast-Link DNA Ligation Kit 

On the Cover: 

EPICENTRE Biotechnologies thanks Dilara Begum for her photo of La Jolla, California. La Jolla (“lah- 

HOY-yah”) is a seaside resort community located 15 minutes from downtown San Diego, California. 

It borders Pacific Beach to the south and extends north to Torrey Pines State Reserve and Del Mar, 

California. La Jolla in Spanish means “The Jewel”, and is home to renowned institutions, such as the 

Scripps Institution of Oceanography, the Salk Institute for Biological Studies, and the Stephen Birch 

Aquarium & Museum. This is in addition to the University of California, San Diego. Furthermore, La Jolla 

is home to many biotechnology and high technology companies. 

Hands on the Cover: 

Heather Meinholz, EPICENTRE Biotechnologies Quality Control 

Volume 14 • Number 2 

www.EpiBio.com 

3


ExactStart Full-Length cDNA Library Cloning Kit: A Rapid and 

Efficient Method to Synthesize Full-Length cDNA for Cloning and 

Accurate Mapping of Transcription Initiation and Polyadenylation Sites 

Introduction 

The ExactStart Full-Length 

cDNA Library Cloning Kit uses a 

novel method to clone full-length, 

mRNA-derived cDNA, synthesized 

from eukaryotic total RNA (FIG 

1). The procedure uses Tobacco 

Acid Pyrophosphatase to hydrolyze 

the phosphoric acid anhydride 

bonds in the triphosphate bridge of 

the cap structure, thereby releasing 

the cap nucleoside and generating 

a 5′-monophosphorylated terminus 

on the RNA molecule. T4 RNA 

ligase is used to add a RNA 

acceptor oligo containing a Not I 

site to the 5′-end of the mRNA. 

A defined 3′ sequence containing 

an Asc I site is then added to 

the cDNA during the oligo(dT)- 

primed reverse transcription of the 

mRNA to cDNA. The resulting 

full-length, first-strand cDNA is 

converted into double-stranded 

cDNA (ds cDNA) and amplified by 

PCR using primers complementary 

to the defined ends of the cDNA. 

The amplified ds cDNA is then 

cloned using the Not I and Asc I 

pre-cut pCDC1 vector included 

in the kit. This approach provides 

a strong selection for full-length 

clones because only 5′-capped and 

3′-poly(A)-tailed mRNA is tagged 

at both ends; RNA types other 

than full-length mRNA will not be 

tagged on both ends, and therefore 

cannot be amplified or cloned into 

the final library. 

Ramesh Vaidyanathan, Les M. Hoffman, and John W. Luecke, EPICENTRE Biotechnologies 

Methods and Results 

The ds cDNA was prepared using total RNA 

isolated from HeLa cells, and analyzed for the 

presence of defined sequences at the 5′- and 

3′-terminal ends using β-actin as a candidate 

mRNA (FIG 2A). To test whether the β-actin 

mRNA is oligo-capped at the 5′-terminal 

end, we performed PCR using primers 

complementary to the oligo adapter and an 

internal primer specific to β-actin. The PCR 

results (FIG 2B, Lane 2) show that a product 

of expected size is produced, suggesting that 

the β-actin mRNA is capped at the 5′-terminal 

end. Similar 3′-terminal analysis shows the 

FIG 1. Schematic of EPICENTRE’s ExactStart Full-Length cDNA Library 

Cloning Kit procedure (see text for details). 

presence of the tag sequence at the 3′-end of 

β-actin mRNA (FIG 2B, Lane 3). 

Thirteen clones were then randomly picked 

from the cDNA library and analyzed for 

insert size by PCR (FIG 3). The results show 

a heterogeneous size distribution of inserts 

ranging in size from 0.35 kbp to > 2.0 kbp. 

Using the ExactStart Full-Length cDNA 

Library Cloning Kit as a discovery tool 

To show the power and benefits provided 

by the ExactStart Kit, cDNA libraries were 

prepared from three eukaryotic model 

organisms: Saccharomyces cerevisiae, 

Schizosaccharomyces pombe, and 

Drosophila melanogaster. Clones 

from the resulting cDNA libraries 

were then sequenced. 

Mapping sites of transcription 

initiation and polyadenylation 

One of the advantages of the 

ExactStart Full-Length cDNA 

Library Cloning Kit is its ability 

to accurately map the sites of 

transcription initiation and 

polyadenylation in an expressed 

gene. The oligo-cap sequence 

present at the 5′-termini of 

cDNAs defines the mRNA start 

site, and the Asc I restriction site 

and oligo dA/dT stretch present 

at the 3′-termini of cDNA marks 

the polyadenylation site. Clones 

were analyzed from a size-selected 

S. cerevisiae cDNA library prepared 

using the ExactStart Kit, and the 

sites of transcription initiation and 

polyadenylation were mapped. All 

of the clones described in FIG 4 are 

full-length and contain the 5′- and 

3′-terminal sequences. 

Novel transcript discovery 

Non size-selected cDNA libraries 

were made from S. cerevisiae, 

S. pombe and D. melanogaster total 

RNA; some of the resulting clones 

were then sequenced. These RNA 

transcripts are almost certainly not 

degradation products or incomplete 

cDNA transcripts due to the 

stringent nature of the ExactStart 

Full-Length cDNA Library Cloning 

Kit’s tagging and amplification 

process. The data show the power of the 

ExactStart Kit to find new and potentially 

significant RNA transcripts. 

Saccharomyces cerevisiae—Several clones 

from the S. cerevisiae library were derived from 

regions in the genome that are not annotated 

as transcriptionally active in the Saccharomyces 

Genome Database (http://www.yeastgenome. 

org/). Some of these unexpected transcriptional 

units, corresponding to antisense, noncoding, 

and intergenic regions, correlate well with 

data obtained from ChIP-Chip microarray 

4 


Volume 14 • Number 2


A. 

5´-end 

B. 

3´-end 

FIG 2. A. Schematic showing the PCR primer map 

positions used for analyzing double stranded HeLa 

cDNA for the presence of the defined sequences at the 

5′- and 3′-termini (see text for details). B. Agarose gel 

analysis of the PCR results; Lane M, mw ladder; Lane 2, 

analysis using a 5′-oligo cap and gene specific primer pair; 

Lane 3, analysis using a gene specific primer and a primer 

for the 3′-tag. 

experiments, indicating RNA polymerase 

occupancy at or near these sites (David Brow, 

University of Wisconsin-Madison, personal 

communication). Furthermore, a number 

of new RNAs were found by cloning the 

“small but not very small” cDNAs (400-500 

bp) without selecting for larger, traditional 

“gene-sized” cDNAs. The repertory of 

“short-ish RNAs” (which we have named 

shishRNAs) varies between organisms. If 

longer (1-3 kbp) cDNA is selected prior to 

cloning, mostly full-length clones of known 

transcriptional units predominate in the 

library. The small and potentially biologically 

relevant transcripts may be missed when larger 

cDNA is selected for cloning. 

i2280709 

FIG 3. PCR analysis of the insert size of thirteen 

cDNA clones produced by the ExactStart Full-Length 

cDNA Library Cloning Kit. Cloning of a non-size 

selected cDNA library can lead to a wide variety of different 

sized cDNA clones. 

Schizosaccharomyces pombe—The S. pombe 

RNA was extracted with EPICENTRE’s 

MasterPure Yeast RNA Purification Kit. We 

found a relatively large number of antisense 

transcripts among the S. pombe cDNA clones. 

About 6% of the total were examined. Two 

of the antisense RNAs were in positions to 

interfere with initiation of transcripts from 

canonical transcription start sites. Around 

60% of the cDNAs encoded ribosomal 

proteins, one-third for the small ribosomal 

subunit and two-thirds for the large subunit. 

Four of the unannotated RNAs contained 

no ORFs coding for more than 40 amino 

acids. The average S. pombe cDNA length was 

470 nt. 

Drosophila melanogaster—Fruitfly S2 cell 

total RNA was obtained from Ambion. About 

55% of the Drosophila cDNA clones encoded 

ribosomal proteins, similar to the fraction of 

S. pombe clones. A very high percentage of the 

cDNAs were apparently full-length. However, 

there was an 11:1 ratio of small subunit to 

large subunit clones. We have no explanation 

for the large imbalance between large and 

small subunit protein cDNA clones. The 

average fruitfly cDNA length was 535 nt. 

Conclusion 

The ExactStart Full-Length cDNA Library 

Cloning Kit can produce full-length cDNA 

libraries from a variety of eukaryotic sources. 

These libraries can be size selected to obtain 

typical full-length cDNAs, which often show 

variations in both 5′- transcriptional start sites 

and 3′-polyadenylation addition sites. If size 

selection is not performed, the ExactStart 

Kit will also clone smaller, unmapped RNA 

molecules, which may have important 

unknown regulatory effects in the cell. 

www.EpiBio.com/exactstart.asp 

NEW! ExactStart Full-Length cDNA Library 

Cloning Kit 

ES0907 

10 Reaction Kits 

www.EpiBio.com/failsafe.asp 

FailSafe PCR PreMix Selection Kit 

FS99060 60 Units 

Contents: FailSafe PCR Enzyme Mix and all 12 

FailSafe PCR 2X PreMixes. Note: Each PreMix 

volume has been modified to match the Enzyme 

Mix volume. 

FailSafe PCR System with PreMix Choice 

FS99100 100 Units 

Contents: Includes FailSafe PCR Enzyme Mix 

and choice of one FailSafe PCR 2X PreMix 

(see website above for complete listing). 

FailSafe PCR System with PreMix Choice 

FS99250 250 Units 

Contents: Includes FailSafe PCR Enzyme Mix 

and choice of two FailSafe PCR 2X PreMixes 

(see website above for complete listing). 

FIG 4. Clone analysis from a size-selected S. cerevisiae cDNA library prepared using ExactStart Full-Length 

cDNA Library Cloning Kit. Red, 5′- and 3′-terminal sequences; Green, base differences when compared to canonical 

sequence; Blue, standard canonical sequence. 

These results were presented at the 2007 

RNA society meeting and are available online 

on the www.EpiBio.com website under 

the What’s New/New Publications button 

menu (www.EpiBio.com/new_pubs.asp). 



5


Fast Real-Time PCR with a Standard qPCR Thermocycler! 

Haiying Grunenwald, John W. Luecke, and James Murray, EPICENTRE Biotechnologies 

Introduction 

EPICENTRE’s new TAQXpedite GREEN 

Real-Time PCR MasterMix Kit generates 

reproducible fast real-time PCR (qPCR) results 

using a standard real-time thermocycler. The 

TAQXpedite GREEN MasterMix Kit achieves 

these results by using a highly efficient enzyme 

blend of thermostable DNA polymerases with 

an optimized 2X MasterMix solution. The 

MasterMix contains EPICENTRE’s patented 

PCR Enhancer (with Betaine),* which 

improves PCR amplification, even with the 

most difficult templates. 1,2 The data presented 

in this short article demonstrate that the easyto-use 

TAQXpedite GREEN MasterMix Kit 

provides superior results in a fast format. 

Benefits of the TAQXpedite GREEN Real- 

Time PCR MasterMix Kit include: 

• Fast qPCR results with standard qPCR 

instrumentation. 

• An optimized 2X MasterMix. 

• PCR Enhancer (with Betaine). 

• High sensitivity and specificity. 


Fast amplification 

To evaluate the performance of the 

TAQXpedite GREEN Real-Time PCR 

MasterMix Kit, a 10-fold serial dilution of 

human cDNA ranging from 10 ng to 10 pg 

was used as the qPCR template. Each reaction 

included: 1X MasterMix, 12.5 pmole of forward 

and reverse primers, and the specified amount 

of human cDNA. All fast qPCR amplifications 

and analyses were performed using a Bio-Rad® 

iCycler iQ® thermocycler. The PCR cycling 

conditions used were: 35 cycles of 2 seconds 

at 92°C and 10 seconds at 70°C for a 210 bp 

fragment of the human β-actin gene. 

1A 

1B 

FIG 1. qPCR data for the TAQXpedite GREEN Real-Time PCR MasterMix Kit comparing reactions cycled 

immediately after set-up and after incubation for 1 hour at room temperature. Corresponding melt curves indicate 

no primer-dimer formation. 

TAQXpedite GREEN MasterMix Kit was 

tested using experimental conditions where 

hot-start is generally recommended. A second 

set of qPCR reactions was assembled, and 

incubated at room temperature for one 

hour before cycling—a process which would 

normally permit any primer-dimers or nonspecific 

PCR products to form. After cycling, 

melt curve and standard curve analyses were 

performed to determine the specificity and 

efficiency of the PCR reactions. 

FIG 1 displays the qPCR quantification curves 

(and melt curve analyses) for the TAQXpedite 

GREEN MasterMix Kit. Reactions incubated 

for 1 hour at room temperature (FIG 1B) 

achieved similar qPCR results as those cycled 

immediately following set-up (FIG 1A). The 

TAQXpedite GREEN MasterMix Kit displayed 

excellent R 2 and PCR efficiency data. 

The TAQXpedite GREEN MasterMix Kit 

produced similar qPCR results, and with 

similar high PCR efficiency, in only about 

one-third the time as a standard 90-minute 

qPCR reaction. In addition, the TAQXpedite 

GREEN MasterMix Kit produced excellent 

PCR efficiency and correlation factors (R 2 = 

0.95–0.99) between replicate amplifications 

using fast qPCR conditions. 

Excellent PCR efficiency and specificity 

In order to simulate conditions which might 

be encountered in high-throughput qPCR 

using automation, the performance of the 

FIG 2. Data showing the qPCR sensitivity, efficiency, and wide dynamic range produced by the TAQXpedite 

GREEN Real-Time PCR MasterMix Kit (see text for details). The amplification was linear over the entirety of the 

8-fold dilution. 

6 




Excellent sensitivity and wide 

detection range 

To examine the sensitivity and detection 

range of the kit, a serial dilution of Lambda 

DNA ranging from 10 7 to 128 copies was 

produced. Each qPCR reaction included: 

1X TAQXpedite GREEN Real-Time PCR 

MasterMix, 12.5 pmole of forward and 

reverse primers, and the appropriate amount 

of lambda DNA. Cycling conditions were 

30 seconds at 98°C, followed by 35 cycles of 

1 second at 92°C and 6 seconds at 70°C. 

FIG 2 shows that, in as little as 30 minutes, 

the TAQXpedite GREEN MasterMix Kit 

produces excellent qPCR sensitivity and 

efficiency with a wide dynamic range. The 

R 2 values and PCR efficiency indicate that 


accurately determines the copy number 

present over an extremely wide detection 

range, ensuring accurate measurement of even 

low abundance transcripts. 

Conclusion 

The TAQXpedite GREEN Real-Time PCR 

MasterMix Kit enables qPCR reactions 

to be performed in as little as 30 minutes 

using standard qPCR instrumentation. Even 

with faster reaction times, results show that 


produces excellent values for R 2 and PCR 

efficiency while maintaining high sensitivity. 

The same excellent qPCR data were obtained 

after incubating the TAQXpedite GREEN 

reaction mixtures at room temperature for one 

hour prior to thermocycling, demonstrating its 

applicability in high-throughput applications. 

References 

1. Henke, W. et al., (1997) Nucl. Acids Res. 25(19), 

3957. 

2. Abu Al-Soud, W. and Rådström, R. J. Clin. 

Microbiol. 38(12), 4463. 

www.EpiBio.com/taqxpedite_green.asp 

NEW! TAQXpedite GREEN Real-Time PCR 

MasterMix Kit 

TXG70796 96-25 µl Reactions 

TXG707400 400-25 µl Reactions 

Contents: 2X MasterMix with Enzyme Blend, 

Passive ROX Reference Dye, Stabilizer, and 

Nuclease Free Water. 

* 

Use of Betaine for DNA Polymerase Reactions, including, but not 

limited to use for PCR or DNA Sequencing, is covered by, U.S. Patent 

No. 6,270,962, European Patent No. 0742838, German Patent No. 

DE4411588C1 and other issued or pending applications in the U.S. 

and other countries that are either assigned or exclusively licensed to 

EPICENTRE. These products are accompanied by a limited nonexclusive 

license for the purchaser to use the purchased products solely 

for life science research. Contact EPICENTRE for information on 

licenses for uses in diagnostics or other fields. 

Easy and Rapid Extraction of Plant DNA 

Using the QuickExtract Plant DNA Extraction Solution 

Les M. Hoffman and James Murray, EPICENTRE Biotechnologies 

The QuickExtract Plant Solution 

enables the inexpensive processing of one 

to hundreds of samples simultaneously 

without centrifugation, spin columns, 

or the use of toxic organic solvents. 

Introduction 

Currently, there is no simple effective solution 

for high throughput extractions of plant leaf 

DNA. Many methods require multiple steps 

and often more than one expensive microtiter 

dish. Traditional hexadecyltrimethylammonium 

bromide (CTAB) based methods 

are laborious, and kits based on spin columns 

are expensive and not designed for high 

throughput. These methods also produce 

DNA samples that are likely to contain polyphenolic 

compounds and polysaccharides, 

which can cause the DNA to be unsuitable 

for PCR amplification. 

EPICENTRE’s new QuickExtract Plant 

DNA Extraction Solution is a simple, rapid, 

and scalable plant leaf DNA extraction 

method for PCR-based applications, including 

Random Amplification of Polymorphic DNA 

(RAPD) and Short Tandem Repeat (STR) 

analyses end-point PCR, or quantitative PCR. 

The QuickExtract Plant Solution enables the 

FIG 1. RAPD analysis using the QuickExtract Plant 

DNA Extraction Kit. One microliter of undiluted 

QuickExtract Plant DNA from the leaves of various 

species of plants were amplified with the RAPD primer 

UBC-888 (5′-BDB(CA) 7 

–3′), where B=G/T/C and 

D=G/A/T. The PCR products were separated on a 2% 

agarose gel and were visualized by staining with SYBR® 

Gold. Lane M, 100 bp ladder markers; Lane 1, pepper; 

Lane 2, soybean; Lane 3, spelt. 

inexpensive processing of one to hundreds 

of samples simultaneously without centrifugation, 

spin columns, or the use of toxic 

organic solvents. It is also an excellent option 

for high throughput applications, and is 

completely compatible for use with robotic 

systems. The 8-minute, 1-tube protocol 

requires no freezing, grinding, or bead beating 

and yields PCR-ready plant DNA after two 

short heating steps. 

The QuickExtract Plant Solution has been 

successfully used to extract DNA from a wide 

range of plant species including corn, soybean, 

spelt, pepper, rosemary, grape, spinach, 

hopvine and Arabidopsis. 

Methods 

DNA was extracted from freshly cut 3-5 

mm leaf discs of spelt, soybean, pepper, and 

spinach. Each leaf disc was placed in an 

individual 500 µl tube or microtiter plate, and 

100 µl of the QuickExtract Plant Solution was 

added to immerse the leaf sample. Care was 

taken not to grind or otherwise damage the 

leaf tissue as this may release contaminating 

polyphenolic compounds. The samples were 

then incubated at 65°C for 6 minutes, 98°C 

for two minutes, and then placed on ice. 

PCR amplification 

End-Point PCR—PCR was carried out 

using EPICENTRE’s FailSafe PCR PreMix 

Selection Kit. This kit contains 12 different 

PreMixes which enables the rapid optimization 

of PCR primers in a single PCR run. One 

microliter from each sample of undiluted 

QuickExtract Plant DNA was added to each 

of the 12 PCR FailSafe PreMixes along with 

Continued on Page 8 



7


Easy Extraction of Plant DNA - Continued from Page 7 

FIG 2. PCR of a single-copy gene using 

QuickExtract Plant DNA Extraction Kit template 

DNA. One microliter of spinach leaf DNA was obtained 

from four different spinach leaves using the QuickExtract 

Plant Solution. The PCR products were separated on a 

2% agarose gel and visualized by staining with SYBR® 

Gold. Lanes 1-4, spinach DNA from four leaves; Lane 5, 

100 bp ladder; Lane 6, no DNA negative control. 

primers and the FailSafe PCR Enzyme Mix. 

The DNA was denatured at 95° for 2 min, 

and then PCR amplified for 40 cycles, each 

consisting of 95°C for 30 sec, 50°C for 10 sec, 

72°C for 45 sec, followed by 72°C for 2 min. 

The PCR products were then separated on a 

2% agarose gel and visualized by staining with 

SYBR® Gold. 

Real-Time PCR—Leaf samples were processed 

in the same manner as for end-point PCR. 

The QuickExtract Plant DNA extracts were 

then serially diluted, and, along with negative 

control samples, amplified using the FailSafe 

GREEN Real-Time PCR System. 

Results 

DNA was extracted from spelt, soybean 

and pepper using the QuickExtract Plant 

Solution. DNA from all three samples was 

successfully used for RAPD analysis as shown 

in FIG 1. PCR on single-copy genes from 

four different samples of spinach DNA clearly 

demonstrated the high quality and uniformity 

of the extracted DNA (FIG 2). 

The QuickExtract Plant DNA samples were 

also shown to work with real-time PCR 

techniques (FIG 3 & 4). The QuickExtract 

Plant Solution provided sufficient template 

DNA for good PCR results even when the 

DNA sample was diluted 1000-fold. With 

this efficient DNA extraction, even very small 

samples can provide enough template DNA for 

hundreds or thousands of qPCR reactions. 

Conclusion 

The QuickExtract Plant DNA Extraction 

Solution represents a rapid and efficient way to 

extract PCR-ready plant genomic DNA from 

most plant samples. The 8-minute, 1-tube 

protocol enables the inexpensive processing 

of one to hundreds of samples simultaneously 

without centrifugation, spin columns, or 

toxic organic solvents. The procedure is fully 

compatible with robotic automation, and 

produces highly reproducible PCR results. 

www.EpiBio.com/quickextract_plant.asp 

NEW! QuickExtract Plant DNA 

Extraction Solution 

QEP70750 

50 ml 

Bulk solution, sufficient to perform 500 100 µl 

extractions. 

www.EpiBio.com/failsafe.asp 

FailSafe PCR PreMix Selection Kit 

FS99060 

60 Units 

Contents: FailSafe PCR Enzyme Mix and all 12 

FailSafe PCR 2X PreMixes. 

www.EpiBio.com/failsafegreen.asp 

FailSafe GREEN Real-Time 

PCR Optimization Kit 

FSR0360 96-25 µl Reactions 

Contents: FailSafe PCR Enzyme Mix, 12 

FailSafe GREEN Real-Time PCR 2X PreMixes, 

and Passive Reference Dye. 

—— no dilution 

—— 1:10 

—— 1:100 

—— 1:1,000 

FIG 3. Real-time PCR using spinach seed 

DNA extracted using the QuickExtract 

Plant DNA Extraction Kit. The target 

amplicon is a 520 bp region of the single-copy 

chromosomal HSC70 gene. The results show 

clear amplification at a thousand-fold dilution 

of the QuickExtract Plant DNA sample. 

Negative controls showed no amplification 

(data not shown). 

—— no dilution 

—— 1:10 

—— 1:100 

—— 1:1,000 

FIG 4. Melting point differential plot for 

real-time PCR of the spinach leaf HSC70 

Gene. The congruence of the melting curves 

indicates that the real-time fluorescence was 

derived from the expected amplicon. 

8 



Evaluation 

Kits are now 

available! 

Detect More Genes Using Your 

Illumina ® Expression BeadChips 

TargetAmp Nano-g Biotin-aRNA Labeling Kit 

for the Illumina ® System 

The TargetAmp Nano-g Biotin-aRNA Labeling Kit for the Illumina® System produces Biotin-aRNA (also 

called cRNA) for transcription profiling using the Illumina® gene expression system such as the Illumina® 

Expression BeadChips. The Biotin-aRNA produced by a TargetAmp Nano-g Kit reaction from 100 ng 

of input total RNA has been shown to detect more genes (RNA transcripts) than Biotin-aRNA produced by 

another kit using 400 ng of input total RNA. 

Benefits 

j Produces Biotin-aRNA that detects more genes using 

less input total RNA than another kit (Fig 2). 

j Produces microgram amounts of Biotin-aRNA with high 

signal intensity from as little as 25 ng of input total RNA. 

j Easy 6-hour procedure yields Biotin-aRNA ready for 

hybridization. 

j Linear RNA amplification process preserves the relative 

transcript abundance of the sample. 

Table 1. The TargetAmp Nano-g Biotin-aRNA Labeling Kit for the 

Illumina® System produces microgram amounts of Biotin-aRNA (also called 

cRNA) from as little as 25 ng of input total RNA. Results summarize experiments 

performed using the HeLa Total RNA control pro vided in the kit. 

Input HeLa Control Total RNA 

Biotin-aRNA Yield 

25 ng 3.1 µg (3100 ng) 

50 ng 5.3 µg (5,300 ng) 

100 ng 10.8 µg (10,800 ng) 

500 ng 75.6 µg (75,600 ng) 

FIG 2. Biotin-aRNA (also 

called cRNA) produced by a 

TargetAmp Nano-g BiotinaRNA 

Labeling Kit for the 

Illumina® System reaction 

detects more genes using less 

input total RNA than BiotinaRNA 

produced by another 

kit. Biotin-aRNA produced 

from 100 ng of total RNA using 

the TargetAmp Nano-g Kit 

and hybridized to the Illumina® 

MouseRef-8 Expression BeadChip 

detected 1656 transcripts that 

were not detected by BiotinaRNA 

produced from 400 ng of 

total RNA using another kit. 

Data courtesy of Dr. Seth Crosby, 

Director for Translational 

Medicine, Washington University 

School of Medicine. 

1656 7896 268 

Input RNA 

100 ng 

• EPICENTRE 

TargetAmp Nano-g 

Biotin-aRNA Labeling Kit 

for the Ilumina ® System 

Input RNA 

400 ng 

• Another 

Supplier’s Kit 

i2260807 

FIG 1. The TargetAmp 

Nano-g Biotin-aRNA 

Labeling Kit for the Illumina® 

System utilizes a linear RNA 

amplification and labeling 

process that yields purified 

Biotin-aRNA in 6 hours. 

Ordering Information 

TargetAmp Nano-g Biotin-aRNA Labeling Kit for the Illumina ® System 

TAN07924 

24 Reactions 

Find more details using the QuickInfo code in website search box! Go to www.EpiBio.com & use 

code: TNDX1

Evaluation 

Kits are now 

available! 

Prepare Biotin-aRNA (cRNA) From as 

Little as 5 Cells for Microarray Studies 


The TargetAmp 2-Round Biotin-aRNA Amplification Kit 3.0 is the only kit that can produce microgram 

amounts of Biotin-aRNA (also called cRNA) from as little as 5 cells (about 50 pg of total RNA) for microarray 

studies. 

Benefits 

j The only RNA amplification kit capable of 

producing micrograms amounts of Biotin-aRNA 

from as little as 5 cells (about 50 pg of input total 

RNA). 

j Biotin-aRNA produced can be hybridized 

to Affymetrix® GeneChip® arrays, Illumina® 

Expression BeadChips and other commercial and 

spotted arrays that utilize Biotin-labeled target 

aRNA. 

j Linear RNA amplification process that preserves 

the relative transcript abundance of the original 

sample. 

j Two-day reaction procedure. 

Table 1. The TargetAmp 2-Round Biotin-aRNA Amplification Kit 3.0 

produces high sensitivity Biotin-aRNA (also called cRNA) from minute 

samples for microarray studies. Biotin-aRNA was prepared from the specified 

amount of mouse liver and skeletal muscle total RNAs using the TargetAmp 

2-Round Biotin-aRNA Amplification Kit 3.0 and then hybridized to the 

Illumina® MouseRef-8 BeadChip array in duplicates. Data were imported into 

BeadStudio (Illumina) for analysis. Genes called “detected” are those with 

detection p-value >0.05. 

Mouse liver 

total RNA 

Mouse skeletal 

muscle 

total RNA 

Input total RNA 50 pg 100 pg 50 pg 100 pg 

Biotin-aRNA Yield 19.3 µg 35.4 µg 12.1 µg 24.2 µg 

Genes (transcripts) detected 7481 8840 7593 8352 

Additional TargetAmp aRNA Amplification Kits 

that produce Biotin-aRNA, aminoallyl-aRNA, and 

unlabeled-aRNA are available. Visit www.EpiBio.com/ 

targetamp.asp for more information. 



TAB2R71010 

10 Reactions 

TAB2R71024 

24 Reactions 


code: RBDX1

In Vitro Transposomics Tools 

Plasmid rescue 

The EZ-Tn5 Insertion Kit is ideal 

for the recovery and propagation of plasmids or other 

circular DNAs that do not contain an E. coli origin of 

replication. The EZ-Tn5 transposon will randomly insert 

into the plasmid DNA, inserting a kanamycin resistance 

marker and the uncommon E. coli plasmid origin R6Kg. 

The plasmid can then be recovered and propagated in 

either the TransforMax EC100D pir + or pir-116 cells, 

depending on the plasmid copy number desired. 

Protein Domain & Epitope Mapping 

The EZ-Tn5 In-Frame Linker Insertion Kit is 

designed to randomly insert 57 base-pair (19 codon) 

insertions into any cloned DNA. The insertions can 

be translated in all three reading frames, and retain 

the original DNA sequence on both sides of the 

insertion. This approach is easier and more versatile 

than traditional linker scanning mutagenesis. 

pir + 

pir-116 

M 1 2 3 4 5 6 7 8 

kb 

10 – 

Plasmid 

DNA 

3 – 

FIG 1. A plasmid containing an EZ-Tn5 

Transposon can be maintained 

in TransforMax EC100D pir + cells at ~15 copies 

per cell (Lanes 1-4) or in TransforMax EC100D 

pir-116 cells at ~250 copies per cell (Lanes 5-8). 

i770411 


EZ-Tn5 Insertion Kit 

EZI011RK 

10 Reactions 

TransforMax EC100D pir + 

Electrocompetent E. coli 

ECP09500 5 x 100 µl 

EZ-Tn5 In-Frame Linker Insertion Kit 

EZI04KN 

10 Reactions 

TransforMax EC100D pir-116 


EC6P095H 5 x 100 µl 


code: EZDX1

In Vitro Transposomics Tools 

EZ-Tn5 Promoter 

Insertion Kit 

The EZ-Tn5 Promoter Insertion Kit is 

designed to randomly insert the T7 phage promoter into 

any DNA. After insertion, RNA transcripts can be made 

using in vitro transcription systems, or in vivo expression 

studies can be performed using bacterial strains containing 

an inducible T7 RNA polymerase gene. 

EZ-Tn5 Transposon 

Construction Vectors 

Create your own in vitro transposon tools with 

our collection of pMOD vectors. These vectors, 

which supply the mosaic ends (MEs) needed 

for transposition, contain a variety of origins of 

replication and antibiotic resistance markers useful 

for the construction of custom research tools. 

Table 1. Replication origins in EZ-Tn5 Transposon Construction 

Vectors. 

EZ-Tn5 Transposon 

Construction Vectors 

ori that is located on 

vector outside of the ME 

sequences 

ori that is located 

within the ME 

sequences 

pMOD-2 colE1 None 

pMOD-3 colE1 R6Kgori 

pMOD-4 R6Kgori None 

pMOD-5 None R6Kgori 

pMOD-6* colE1 None 

*Contains a Kan R marker inside of the ME 


EZ-Tn5 pMOD Transposon 

Construction Vectors: 


-2 

MOD0602 20 µg 

-3 

MOD1503 20 µg 

-4 

MOD4804 20 µg 

-5 

MOD4805 20 µg 

-6 

MOD7906 20 µg 

Transposon Construction Vectors include Forward 

and Reverse PCR Primers. 

EZ-Tn5 Promoter Insertion Kit 

EZI03T7 10 Reactions 

EZ-Tn5 Transposase 

TNP92110 10 Units 


code: EZDX1

PCR-ready Plant Genomic DNA 

in 8 minutes 


The QuickExtract Plant DNA Extraction Solution can be used to rapidly and efficiently extract PCR-ready genomic DNA 

from most plant samples using a simple 1-tube protocol that takes only 8 minutes. Most leafy plants are acceptable for DNA 

extraction using the QuickExtract Plant Solution, including 

Arabidopsis, spelt, corn, soybeans, spinach, and pepper leaves 

(FIG 1). 

The QuickExtract Plant method allows for the inexpensive 

processing of one to hundreds of samples simultaneously– 

without centrifugation, spin columns or use of any toxic 

organic solvent. The procedure is fully compatible with 

robotic automation, provides a PCR-ready sample, and 

has been proven reproducible (FIG 2). Simply add the 

QuickExtract Plant Solution to the sample and perform two 

sequential heating steps. A small aliquot of the sample mix is 

then used as a template for PCR or qPCR reactions. 

Add Plant QuickExtract 

DNA Extraction Solution 

to sample 

Heat at 65ºC for 6 minutes 

and 98ºC for 2 minutes 

PCR-ready 

DNA 

i2240707 

Applications 

j Plant Genomic studies. 

j High throughput plant applications. 

j GMO testing. 

Benefits 

j PCR-ready sample. 

j No bead beating, freezing or grinding of plant leaf material. 

j Nontoxic, inexpensive processing. 

j Fast procedure (8 min for average sample). 

j No centrifugation. 

j Multiple samples can easily be processed simultaneously. 

FIG 1. PCR products using QuickExtract Plant DNA 

Extraction Solution with different varieties of plant leaves. 

40 cycles of RAPD with primer UBC-866 and 1 µl of Plant 

QuickExtract DNA preps from leaves with FailSafe PCR 

System. Lane M, 100 bp ladder; Lane 1, Pepper; Lane 2, 

Soybean and Lane 3, Spelt. 

FIG 2. Reproducible PCR Results with 

QuickExtract Plant on Arabidopsis 

thaliana leaves. Six individual punches 

of four Arabidopsis leaves were treated 

with QuickExtract Plant Solution. 

One microliter of the solution was used 

in a 25 µl PCR with the optimized 

PreMix of the FailSafe PCR System 

and primers specific for the single-copy 

HSC70 chromosomal gene. After thirty 

five cycles of amplification, aliquots 

were electrophoresed in a 2% agarose gel 

and the DNA visualized by staining with SYBR Gold. Lanes 1-6, leaf punch 

QuickExtract DNAs; Lane 7, no leaf negative control; Lane 8, 100 bp ladder 

DNA markers. The expected amplicon is approximately 520 bp. 



QEP70750 

50 ml 

Bulk solution, sufficient to perform 500 100 µl extractions. 


code: QPDX1

PCR-ready Genomic DNA 

in 3 - 8 minutes 


The QuickExtract DNA Extraction Solution 1.0 can be used to rapidly and efficiently extract PCR-ready genomic DNA 

from almost any sample type using a simple 1-tube protocol that takes only 3 - 8 minutes, depending on the sample (FIG 1). 

Common samples for DNA extraction using QuickExtract Solution include hair follicles, quill-end cells of feathers, tissue culture 

cells, buccal cells, and mouse tail snips. It is useful for extracting PCR-ready DNA in high yield for genomic, transgenic, or viral 

DNA screening in animals, or for genetic or environmental research and screening in humans or other organisms. 

The QuickExtract method allows for the inexpensive processing of one to hundreds of samples simultaneously, without centrifugation, 

spin columns or the use of any toxic organic solvent. The method is also compatible with robotic automation (FIG 2). 

QuickExtract DNA Extraction Solution is also used in the BuccalAmp DNA Extraction 

Kit using a 3-minute single-tube protocol for extraction of PCR-ready DNA from buccal 

cells. The method permits the use of samples obtained by non-invasive means to collect 

buccal cells with our Catch-All foam swab. The BuccalAmp kit is supplied with our 

Catch-All swabs and ready to use 500 µl aliquot tubes of QuickExtract solution. 

Tail 

Snips 

Feathers 

Hair 

Follicles 

Applications 

j Simple, rapid extraction of PCRready 

DNA. 

j Transgenic mouse genotyping. 

j Genetic studies. 

j Human identity testing. 

j Viral or microbial screening. 

Benefits 

j Nontoxic, inexpensive processing. 

j Short procedure. 

j No centrifugation. 

j High throughput and robotic 

compatible. 

Bacteria 

Add Samples 

QuickExtract 

Solution 

Marker 

Human buccal cells 

Neoplastic cells (HeLa) 

Human hair follicle 

Mouse-tail tissue 

Bacteria 

Transgenic mouse 

Heat at 65ºC for 6 minutes 

and 98ºC for 2 minutes 

M 1 2 3 4 5 6 

FIG 1. FailSafe PCR amplifications of genomic DNA extracted 

from a variety of tissues or cells. Buccal cells are extracted using 

BuccalAmp DNA Extraction Kit, and all others using QuickExtract 

DNA Extraction Solution. Lanes 1-3, human β-globin; Lane 4, transgenic 

mouse GAPDH; Lane 5, E. coli 16S ribosomal RNA gene; Lane 

6, transgenic SV40 T antigen. 

PCR-ready 

DNA 

FIG 2. Procedure for obtaining PCRready 

DNA using QuickExtract DNA 

Extraction Solution. 



QE09050 

50 ml 

Bulk solution, sufficient to perform 100 extractions. 

BuccalAmp DNA Extraction Kits 

BQ0901S 

1 Kit (15 Tubes & Swabs) 

Catch-All Sample Collection Swabs 

QEC091H 

100 Swabs 


code: QEDX1

FAST End-Point PCR with 

a Standard Thermocycler 

TAQXpedite PCR System (FAST End-Point) 

With a standard thermocycler, the TAQXpedite PCR System has been shown to achieve FAST PCR results by 

using enzyme/reagent combinations that improve PCR efficiency. The TAQXpedite PCR System (FAST End- 

Point) contains a unique blend of thermostable DNA polymerases that can be used for FAST PCR reactions. The 

kit contains the enzyme mix with a carefully optimized 2X Universal MasterMix, and a difficult/long MasterMix 

with all four dNTPs, and an optimized MgCl 2 

concentration. The MasterMix also contains EPICENTRE’s 

patented PCR Enhancer (with Betaine), which substantially improves the yield, efficiency, and specificity of 

amplification of many target sequences, especially those containing a high GC content or secondary structure.1-⁵ 

Benefits 

j FAST PCR with a standard PCR instrument. 

j High fidelity. 

j Amplify up to 80% GC content. 

j Fast PCR for amplicons longer than 30 kb. 

j FAST multiplexing. 

j Two convenient MasterMixes for universal and 

difficult/long amplifications. 

j Fully licensed for PCR research applications. 

References 

1. Schanke, J.T. and Grunenwald, H.L. (1997) EPICENTRE Forum 4 (1), 2. 

2. Grunenwald, H.L. and Schanke, J.T. (1997) EPICENTRE Forum 4 (1), 4. 

3. Mytelka, D.S. and Chamberlin, M.J. (1996) Nucl. Acids Res. 24, 2774. 

4. Henke, W. et al., (1997) Nucl. Acids Res. 25, 3957. 

5. Weissensteiner, T. and Lanchbury, J.S. (1996) BioTechniques 21, 1102. 

FIG 1. Amplification 

using Lambda DNA 

as a starting template 

for a 539 bp amplicon. 

Different starting 

concentrations of template 

were used in a 16 minute 

reaction. Lane 1, kb 

ladder; Lane 2, 100 pg; 

Lane 3, 10 pg; Lane 4, 1 

pg; Lane 5, kb ladder. 

FIG 2. A comparison 

of TAQXpedite PCR 

System, Competitor Q, and 

Competitor A, using FAST 

PCR to amplify a 4 kb 

fragment. Gel image shows 

the results starting with 1 ng 

Lambda DNA as the template 

for the PCR reaction. Lane 1, 

MW; Lane 2, TAQXpedite; 

Lane 3, Competitor Q’s Fast 

Kit; and Lane 4, Competitor 

A’s Fast PCR MasterMix. 


TAQXpedite PCR system (FAST End-Point) 

TXP78200 

200 25-µl Reactions 

TXP78001 

1,000 25-µl Reactions 


code: TEDX1

FAST Real-Time PCR with 

a Standard qPCR Thermocycler 

TAQXpedite GREEN Real-Time PCR MasterMix Kit 

Using a standard Real-Time thermocycler, the TAQXpedite GREEN Real-Time PCR MasterMix Kit is able 

to achieve FAST PCR results by using an enzyme/reagent combination to improve the PCR efficiency. The 

TAQXpedite GREEN Kit b includes a unique blend of thermostable DNA polymerases with an optimized 

2X MasterMix solution containing all four dNTPs, MgCl 2 

and SYBR® Green I dye. The MasterMix also 

contains EPICENTRE’s patented PCR Enhancer (with Betaine a ), which substantially improves the yield 

and specificity of amplification of many target sequences, especially those containing a high GC content or 

secondary structure. 1-5 

Benefits 

j FAST reproducible qPCR. 

j High sensitivity and specificity. 

j Wide dynamic range with excellent PCR efficiencies. 

j Convenient 2X MasterMix, plus all reaction compounds 

included in kit. 

j Fully licensed for qPCR research applications. b 

References 

1. Schanke, J.T. and Grunenwald, H.L. (1997) EPICENTRE Forum 4 (1), 2. 

2. Grunenwald, H.L. and Schanke, J.T. (1997) EPICENTRE Forum 4 (1), 4. 

3. Mytelka, D.S. and Chamberlin, M.J. (1996) Nucl. Acids Res. 24, 2774. 

4. Henke, W. et al., (1997) Nucl. Acids Res. 25, 3957. 

5. Weissensteiner, T. and Lanchbury, J.S. (1996) BioTechniques 21, 1102. 

FIG 1. Serial dilutions of Lambda DNA from 10 7 to 128 copies were 

amplified. Each real-time PCR reaction included 1X MasterMix, 12.5 pmole 

each of forward and reverse primers and template. Cycling conditions were 

30 seconds at 98°C, followed by 25 cycles of 1 second at 92°C and 6 seconds at 

70°C. This serial dilution demonstrates a consistent qPCR reaction and a 

reaction efficiency of 103.9%. 


TAQXpedite Ordering Information 

GREEN Real-Time PCR MasterMix Kit 

TXG70796 


TXG707400 



code: TGDX1 

SYBR is a registered trademark of Molecular Probes, Inc., Eugene, Oregon. Tween is a registered trademark of ICI Americas Inc., Wilmington, Delaware. TAQXpedite, MasterAmp, MasterPure and FailSafe are 

trademarks of EPICENTRE, Madison, Wisconsin. SYBR Green I Dye is covered by issued and pending patents owned by Molecular Probes, Inc., Eugene, OR. Products containing SYBR Green Dye are licensed by 

EPICENTRE for use in enzyme-based nucleic acid amplification procedures, including real-time PCR, solely for life science research. 

a 

Use of Betaine for DNA Polymerase Reactions, including, but not limited to use for PCR or DNA Sequencing, is covered by, U.S. Patent No. 6,270,962, European Patent No. 0742838, German Patent No. 

DE4411588C1 and other issued or pending applications in the U.S. and other countries that are either assigned or exclusively licensed to EPICENTRE. These products are accompanied by a limited non-exclusive 

license for the purchaser to use the purchased products solely for life science research. Contact EPICENTRE for information on licenses for uses in diagnostics or other fields. 

b 

Covered by U.S. Patent No. 5,436,149 and other patents pending worldwide, owned by Takara Shuzo, Co., Ltd. and licensed to EPICENTRE. 

EPICENTRE’s PCR products are sold under licensing arrangements with F. Hoffmann-La Roche Ltd., Roche Molecular Systems, Inc., and Applied Biosystems. The products containing a thermostable DNA 

polymerase are accompanied by a limited license to use it in the Polymerase Chain Reaction (PCR) and RT-PCR for life science research in conjunction with a thermal cycler whose use in the automated performance 

of the PCR process is covered by the up-front license fee, either by payment to Applied Biosystems or as purchased, i.e., an authorized thermal cycler. Go to www.EpiBio.com for complete license statements.


Questions about RNA capping and in vitro transcription 

Q. What is RNA capping 

A. Capping is the first step in the maturation 

of eukaryotic mRNA. Shortly after the start 

of transcription, the 5′-end of the growing 

RNA molecule is capped by the addition of 

a guanosine monophosphate residue (from 

GTP) via a 5′-5′ pyrophosphate linkage. After 

addition of the cap nucleotide, a methyl group 

is added to the N7 position of the guanine 

base producing a Cap 0 structure. 

Q. What modifications to in vitro transcribed 

RNA are required for efficient translation of 

the RNA in vivo 

A. For in vitro-transcribed RNA to be 

translated efficiently in vivo, the mRNA 

requires two things: 

• A poly(A)-tail at the 3′-end of the transcript. 

• A correctly orientated N7-methylated cap 

(Cap 0) at the 5′-end of the molecule. 

However, the addition of another methyl 

group onto the penultimate nucleotide from 

the 5′-end of the mRNA (producing a Cap 1 

structure) will further boost translation. 

Q. How much better are the translation 

efficiencies if I use RNA with a Cap 1 

structure instead of a Cap 0 structure 

A. It has been reported that methylation 

at the 2′-O position of the penultimate 

nucleotide of capped RNA (to give a Cap 1 

structure) improves translation by 20% to 

50% over RNA with a Cap 0 structure.1,2 

Q. Which process has better capping 

efficiency, EPICENTRE’s new mScript 

mRNA Production System or cap analog-based 

co-transcriptional systems 

A. The mScript mRNA Production System 

is the first commercially available kit that 

produces capped mRNA with nearly 100% 

efficiency, and with all of the caps in the 

correct orientation.3 Direct incorporation of 

cap analog dinucleotides during traditional in 

vitro co-transcriptional systems is considerably 

less efficient, with capping efficiencies 

approaching only 75-80%. In addition, with 

some cap analogs, 30% or more of the 

caps are incorporated into mRNA in the 

wrong orientation, rendering such mRNA 

untranslatable. 

Q. Why is there such a large improvement 

in RNA capping efficiency using the mScript 

mRNA Production System when compared to 

cap analog-based co-transcriptional reactions 

A. Unlike traditional cap analog-based 

co-transcriptional reactions that are both costly 

and inefficient, the mScript System uses an 

enzymatic process to build cap structures onto 

the RNA transcripts. Direct incorporation 

of cap analog during in vitro transcription is 

inconsistent in its capping efficiency and cap 

orientation. There is also an inherent trade-off 

between capping efficiency and total RNA yield 

from the transcription reaction; samples with 

high capping efficiencies produce lower overall 

RNA yields. The mScript System includes 

the Vaccinia virus-derived capping enzyme, 

which contains all three enzymatic activities 

(mRNA triphosphatase, guanylyltransferase, 

and guanine-7-methyltransferase) necessary 

to build 5′-Cap 0 structures in vitro, ensuring 

nearly 100% capping with 100% proper cap 

orientation. Furthermore, the mScript System 

contains the Vaccinia 2′-O-Methyltransferase 

enzyme, allowing the natural, translationboosting 

Cap 1 structure to be built. 

Q. Will EPICENTRE’s ScriptCap system 

work with all in vitro transcribed RNA 

A. Yes. ScriptCap Capping Enzyme will work 

on any in vitro transcribed RNA provided that 

the RNA has a 5′ di- or tri-phosphate. 

Q. What is the main benefit of using Anti- 

Reverse Cap Analog (ARCA) dinucleotides as 

opposed to standard cap analog dinucleotides 

for capping of RNA by co-transcriptional 

incorporation 


A. Standard cap analogs can be incorporated 

at the 5′-end of the RNA in both the forward 

[m 7 G(5′)ppp(5′)G(pN)...] and the reverse 

orientation [G(5′)ppp(5′)m 7 G(pN)...]; RNA 

molecules with the cap in the reverse orientation 

are not efficiently translated. To overcome this 

problem, EPICENTRE offers an Anti-Reverse 

Cap Analog. This cap analog has a 3′-OCH 3 

group on one of the nucleotides, so the ARCA 

will be incorporated by the RNA polymerase to 

produce RNA transcripts capped exclusively in 

the correct orientation. 

Q. Which capping system is right for my 

needs 

A. Generally, mRNA produced using the 

mScript mRNA Production System will 

outperform the RNA produced using a cap 

analog during in vitro transcription. However, 

cap analog systems such as the AmpliCap 

and MessageMAX kits may have some 

advantages for certain applications, see the 

table below. 

AmpliCap-MAX 

T7 and T3 High 

Yield Message 

Maker Kits 

MessageMAX 

T7 Capped 

Message 

Transcription Kit 

- ARCA-Capped 

mScript 

mRNA 

Production 

System 

Reaction time 30 min 30 min 2 hrs 

Capping efficiency ~50* ~80% ~100% 

Reverse 

Yes No No 

incorporation of cap 

Yield per reaction 60 µg 60 µg 60 µg 

Capping of difficultto-cap 

Yes Yes Varies 

transcripts # 

Poly(A) Polymerase No † No † Yes 

included 

2′-O-Methyltransferase No ‡ No ‡ Yes 

included 

*Using a typical 4:1 ratio of dinucleotide cap analog to GTP, only about 

80% of the transcripts are capped, of which about 60% are in the 

correct orientation. 

# 

These include transcripts with extremely strong 5′ hairpin structures. 

Contact Technical Services for more details. 

† 

The A-Plus Poly(A) Polymerase Tailing Kit can be purchased 

separately. 

‡ 

The ScriptCap 2′-O-Methyltransferase can be purchased separately. 

References 

1. Kuge, H. et al., (1998) Nucl. Acids Res. 26(13), 

3208. 

2. Meis, R. and Meis, J.E. (2006) EPICENTRE 

Forum 13(4), 5. 

3. Meis, J.E. and Meis, R. (2007) EPICENTRE 

Forum 14(1), 4. 


17


A Simple Enzymatic Process for Generating mRNA-Enriched 

Poly(A)-Tailed RNA from Total RNA Samples 

Merriann Carey, EPICENTRE Biotechnologies 

FIG 1. Overview of the mRNA-ONLY Prokaryotic 

mRNA Isolation Kit with Poly(A)-Tailing. 

Terminator Exonuclease digests the 16S and 23S 

rRNAs in prokaryotic RNA to yield an enriched mRNA 

preparation. The remaining RNA molecules are then 

polyadenylated at the 3′-end using A-Plus Poly(A) 

Polymerase in the presence of ATP. 

Introduction 

Metagenomics has spurred a growing 

interest in using environmental mRNA or 

transcriptomes to correlate genetic potential 

with patterns of microbial activity. However, 

progress has been hindered in part by the 

difficulties of working with bacterial mRNA. 

Bacterial mRNA generally lacks the poly(A) 

tail, which makes the isolation and analyses 

of eukaryotic messages fairly straightforward. 

Moreover, bacterial rRNAs, like their 

eukaryotic counterparts, can make up 80% 

or more of total RNA, overwhelming samples 

with background signals. EPICENTRE’s new 

mRNA-ONLY Prokaryotic mRNA Isolation 

Kit with Poly(A)-Tailing overcomes these 

limitations with a simple two-step enzymatic 

process that generates mRNA-enriched 

poly(A)-tailed RNA from total cellular RNA 

samples. The resulting polyadenylated mRNA 

can then serve as a template for synthesizing 

cDNA for quantitative RT-PCR, cDNA 

library construction, and array analysis. 

Production of mRNA-enriched 

poly(A)-tailed RNA 

The two-step enzymatic process provided 

by the mRNA-ONLY Prokaryotic mRNA 

Isolation Kit with Poly(A)-Tailing is shown 

in FIG 1. EPICENTRE’s Terminator 

5′-Phosphate-Dependent Exonuclease* is 

used to digest ribosomal RNAs having a 

5′-monophosphate. This processive 5′–3′ 

exonuclease does not digest RNA having a 

5′-triphosphate, a 5′-cap structure (present on 

most eukaryotic mRNAs), or a 5′-hydroxyl 

group. Consequently, Terminator Exonuclease 

can be used to isolate prokaryotic mRNA with 

minimal carryover of 16S and 23S rRNA. 1 

The remaining RNA molecules are then 

polyadenylated at the 3′-end using A-Plus 

Poly(A) Polymerase in the presence of ATP. 

First-Strand cDNA Synthesis 

As a simple test case, mRNA-enriched 

poly(A)-tailed E. coli RNA, prepared using 

the mRNA-ONLY Prokaryotic mRNA 

Isolation Kit with Poly(A)-Tailing, was reverse 

transcribed using an oligo(dT) primer and 

MMLV Reverse Transcriptase (MMLV RT). 

The resulting cDNA was used for real-time 

PCR (qPCR) without additional treatment 

or purification. qPCR was performed using 

the TAQurate GREEN Real-Time PCR 

MasterMix. a Control reactions were also 

performed under the same conditions except 

that MMLV RT was omitted. 

FIG 2. Analysis of mRNA-enriched poly(A)-tailed 

E. coli RNA by real-time RT-PCR. Each amplification 

plot is color coded and the corresponding gene designations 

are listed in Table 1. 

Target ETCN C T 

fliC 51 17.2 

rplK 51 17.9 

pal 18 18.1 

cyoA 16 20.6 

ilvE 4 22.0 

basR 2 22.4 

fecD 0.3 25.9 

yafL 0.1 27.0 

Table 1. Comparison of 

Estimated Transcription 

Copy Number (ETCN) 

and Cycle Threshold (C T 

) 

Values. 

As shown in FIG 2 and Table 1, the average 

cycle threshold (C T 

) value for each of the 

eight targets tested correlates well with the 

estimated transcription copy number (ETCN) 

derived from E. coli microarray data. 2 Thus, 

a highly expressed gene such as fliC with 

an ETCN of 51 has a correspondingly high 

real-time RT-PCR signal (low C T 

value). Even 

the 3-fold difference in ETCN levels for the 

rare sequences fecD and yafL was represented 

by reproducible differences in C T 

values. 

Preservation of relative mRNA 

abundance levels 

To evaluate the effect of Terminator 

Exonuclease and A-Plus Poly(A) Polymerase 

on the relative abundance of the eight mRNA 

transcripts listed in Table 1, real-time RT-PCR 

was performed before and after treatment with 

these two enzymes. The “untreated” cDNA 

was produced from total E. coli RNA using 

MMLV RT and random nonamer primers. 

The “treated” cDNA was produced as described 

in the previous section. The results shown in 

FIG 3 are expressed as the difference in C T 

values (∆C T 

) between the target message and 

the normalizer (pal) for untreated and treated 

samples. The high correlation coefficient 

of R 2 =0.935 demonstrates that the cDNA 

produced from the mRNA-enriched poly(A)- 

tailed RNA conserved the relative abundance 

of different RNA species. 

Amplification of environmental mRNA 

To validate the efficacy of Terminator 

Exonuclease and A-Plus Poly(A) Polymerase 

in the analysis of environmental RNA, a 

fresh sample of dog feces, known to be rich 

in bacteria, was used. Initially, DNA isolated 

from dog feces (using the MasterPure DNA 

Purification Kit) was screened by end-point 

PCR with the same set of primers used to 

amplify the E. coli genes listed in Table 1. 

Four of the eight PCR reactions (cyoA, pal, 

rplK, and yafL) yielded amplicons of the 

expected size (data not shown). 

Total RNA isolated from the same fecal 

sample as the genomic DNA, was then treated 

sequentially with Terminator Exonuclease and 

A-Plus Poly(A) Polymerase (FIG 4). Since the 

amount of total RNA from environmental 

samples is likely to be small, the poly(A)-tailed 

RNA produced by the Terminator Exonuclease 

and A-Plus Poly(A) Polymerase treatment 

was amplified using the MessageBOOSTER 

cDNA Synthesis Kit for qPCR b before further 

Relative Expression (∆C T ) 

10 

8 

6 

4 

2 

0 

-2 

yafL fecD basR ilvE cyoA rplK fliC 

RNA Target 

Untreated 

Terminator Exonuclease + 

A-Plus Poly(A) Polymerase 

FIG 3. Treatment of E. coli RNA with Terminator 

Exonuclease and A-Plus Poly(A) Polymerase maintains 

relative mRNA abundance levels as determined 

by real-time RT-PCR. 

c1930705 

18 




FIG 4. Denaturing agarose 

gel analysis of total fecal 

RNA before (-) and after (+) 

digestion with Terminator 

Exonuclease. The mRNAenriched 

sample was then 

polyadenylated with A-Plus 

Polymerase (data not shown). 

analysis. MessageBOOSTER reactions 

utilize a linear RNA amplification process 

that produces large amounts of anti-sense 

RNA (aRNA, also called cRNA) from poly(A) 

RNA, and then converts the aRNA to cDNA 

for qPCR. 3 

One hundred picograms of mRNA-enriched 

poly(A)-tailed fecal RNA was used in a 

standard MessageBOOSTER reaction, and 

5 to 15% of the resulting cDNA was then 

used for real-time RT-PCR. As shown in 

FIG 5A, two of the four targets tested (pal 

and yafL) yielded satisfactory qPCR signals. 

The specificity and size of the PCR products 

were verified by melting curve analysis and 

gel electrophoresis (FIG 5, B and C). qPCR 

of cDNA produced from 100 pg of enriched 

and tailed RNA, without the benefit of a 

MessageBOOSTER reaction, failed to detect 

any of these four targets (data not shown). 

Panel A 

Conclusion 

Global analysis of microbial transcripts in 

environmental samples typically relies on the 

conversion of RNA to cDNA via random 

priming. Random priming coupled with the 

abundance of rRNA can overwhelm low 

level transcripts so that their subsequent 

amplification may not be quantitative. The 

specificity of oligo(dT) priming is therefore 

often cited as the best method to use when 

a faithful cDNA representation of the 

mRNA pool is desired. 4 Moreover, oligo(dT) 

priming is the most appropriate choice when 

amplification of mRNAs from limited RNA 

samples is desired. 

The results shown here demonstrate that the 

mRNA-ONLY Prokaryotic mRNA Isolation 

Kit with Poly(A)-Tailing provides a simple 

enzymatic process for generating mRNAenriched 

poly(A)-tailed RNA from total RNA 

samples. The resulting polyadenylated mRNA 

lacks the confounding effects of rRNA and 

makes an ideal template for cDNA synthesis 

using an oligo(dT) primer. 

References 

1. Meis, J. E. and Pease, J. (2006) EPICENTRE 

Forum 13(1), 22. 

2. Liu, M. et al., (2005) J. Biol. Chem. 280(16), 

15921. 

3. Grunenwald, H. et al., (2006) EPICENTRE Forum 

13(1), 7. 

4. Bustin, S.A. et al., (2005) J. of Mol. Endocrinol 

34(3), 597. 

*All products used in this article are from EPICENTRE 

Biotechnologies unless otherwise stated. 

Panel B 

www.EpiBio.com/mrnaonly.asp 

mRNA-ONLY Prokaryotic mRNA Isolation Kit 

with Poly(A)-Tailing 

MOT60510 

10 Reactions 

Contents: Terminator 5′-Phosphate-Dependent 

Exonuclease, mRNA-ONLY 10X Reaction Buffer, 

RNase Inhibitor, mRNA-ONLY Stop Solution, 

5M LiCl Solution, A-Plus Poly(A) Polymerase, 

10X A-Plus Reaction Buffer, 10 mM ATP, and 

RNase-Free Water. 

www.EpiBio.com/mmlv.asp 

MMLV Reverse Transcriptase 

M6125H 10 U/µl 2,500 Units 

M6110K 10 U/µl 10,000 Units 

M4425H 50 U/µl 2,500 Units 

M4410K 50 U/µl 10,000 Units 

Includes 10X Reaction Buffer and DTT. 

www.EpiBio.com/taqurate.asp 

TAQurate GREEN Real-Time PCR MasterMix 

TM049096 96 25-µl Reactions 

TM046400 400 25-µl Reactions 

Contents: TAQurate Real-Time PCR Enzyme 

Blend, TAQurate GREEN Real-Time 2X PCR 

MasterMix, Passive Reference Dye, and Stabilizer. 

www.EpiBio.com/masterpure_complete.asp 

MasterPure DNA Purification Kit 

(for isolating TNA or DNA) 

MCD85201 200 Purifications 

Contents: Red Cell Lysis Solution, Tissue and Cell 

Lysis Solution, MPC Protein Precipitation Reagent, 

2X T&C Lysis Solution, TE Buffer, RNase A, and 

Proteinase K. 

www.EpiBio.com/messagebooster.asp 

MessageBOOSTER cDNA Synthesis Kit 

for qPCR 

MB060110 

10 Reactions 

MB060124 

24 Reactions 

Panel C 

cyoA pal 

M 1 2 3 4 

rplK yafL 

M 5 6 7 8 

a 

EPICENTRE’s PCR products are sold under licensing arrangements 

with F. Hoffmann-La Roche Ltd., Roche Molecular Systems, Inc., 

and Applied Biosystems. The products containing a thermostable 

DNA polymerase are accompanied by a limited license to use it in 

the Polymerase Chain Reaction (PCR) and RT-PCR for life science 

research in conjunction with a thermal cycler whose use in the automated 

performance of the PCR process is covered by the up-front 

license fee, either by payment to Applied Biosystems or as purchased, 

i.e., an authorized thermal cycler. 

b 

MessageBOOSTER is a trademark of EPICENTRE Biotechnologies, 

Madison, WI. This product is covered by intellectual property licensed 

to EPICENTRE Technologies Corporation from Johnson & Johnson 

Pharmaceutical Research & Development, L.L.C. This product is 

covered by U.S. Patents licensed exclusively to Incyte Corporation and 

sublicensed to EPICENTRE Technologies Corporation. See www. 

EpiBio.com website for complete license statements. 

FIG 5. Real-time RT-PCR analysis of mRNA-enriched poly(A)-tailed fecal RNA. Panel A, qPCR graphs for pal and 

yafL with no template controls; Panel B, melt-curve analysis; Panel C, Gel electrophoresis of RT-PCR products from 

E. coli (odd lanes) and fecal (even lanes) samples. 



19


Protein Expression from a Chromosomally-Inserted Transposon 

Darin J. Haskins, EPICENTRE Biotechnologies 

20 

Introduction 

The expression of proteins in microorganisms 

for research or commercial applications is 

accomplished almost exclusively using 

plasmid-based expression systems. An example 

is the bacteriophage T7 expression system 

pioneered by Dr. William Studier,1 which 

is used by many researchers to obtain a 

high-level expression of cloned proteins in E. 

coli. This system utilizes a plasmid in which 

the gene of interest is cloned downstream of 

the phage T7 gene 10 promoter and a host 

strain that is capable of expressing T7 RNA 

polymerase upon induction. 

Although plasmid-based expression systems 

can be very useful, they are not without 

problems. Plasmids can place a large metabolic 

burden on host cells. Therefore, antibiotics 

may be required in the growth medium in 

order to avoid loss of the plasmid during 

cultivation.2 Furthermore, employment of 

a strong plasmid selection method can kill 

cells that lose the plasmid, leading to an 

accumulation of non-viable and lysed cell 

matter, which complicates purification.3 

Overexpression of the cloned gene or other 

genes located on the plasmid can also lead to 

lower plasmid segregation stability.2 

The standard practice of using antibiotic 

selection for plasmid maintenance also limits 

which plasmids and antibiotic resistance genes 

can be used for production of pharmaceutical 

grade proteins. This is because regulatory 

standards do not allow use of certain antibiotics 

because of the potential for patient allergic 

reactions to low levels of residual antibiotics. 

An expression transposon, stably integrated 

into the chromosome of a microorganism, 

can alleviate many problems related to 

protein expression. The transposon can 

be inserted into the host chromosome using 

an EZ-Tn5 Transposome complex 

(EPICENTRE Biotechnologies). In this 

method, the transposase is pre-bound in vitro 

to the 19-base mosaic ends (i.e., transposase 

recognition sequences) of an “artificial” 

transposon in the absence of Mg ++ , and then 

electroporated into the microbe. Once inside 

the cell, this EZ-Tn5 Transposome is activated 

by Mg ++ in the host’s cellular environment, 

where the Transposome efficiently and 

randomly inserts its transposon DNA into 

the genomic DNA of the host cell. The 

transposon is called “artificial” because it lacks 


FIG 1. T7 expression cassettes were engineered into the EZ-Tn5 pMOD-6 Transposon 

Construction Vector in the configuration shown above. 

a gene that encodes the transposase. Therefore, 

the transposon is completely stable and cannot 

excise or reinsert itself into a new position 

on the chromosome.4 Antibiotics or other 

selective pressures are not required to maintain 

the transposon insertion, and if desired, the 

antibiotic resistance marker used to select for 

the initial insertion can be removed by standard 

genetic engineering methods. 

Any DNA can be used to make an 

EZ-Tn5 transposon simply by adding 

transposase recognition sequences to both 

ends. Here, we report construction and use 

of EZ-Tn5 Transposomes for expression 

of two different proteins from the E. coli 

chromosome. 


Protein over-expression 

T7 expression cassettes were engineered into 

the new EZ-Tn5 pMOD-6 Transposon Construction Vector in 

the configuration seen in FIG 1. Briefly, 

transcription of the gene to be expressed is 

driven by a T7 promoter located upstream 

of the open reading frame. The kanamycin 

resistance gene is included in the construct to 

provide a marker for the initial insertion event 

of the Transposome DNA into the E. coli 

chromosome. It is not necessary to utilize this 

marker in subsequent culturing, and it can be 

removed post-insertion if desired. 

The Transposome complex was created by 

cutting the protein expression cassette from 

the pMOD vector using the Pvu II restriction 

sites outside of the mosaic ends. Alternatively, 

transposon DNA can be generated by 

standard PCR as described in EPICENTRE’s 

product literature (www.EpiBio.com). The 

transposon DNA was then incubated with 

EZ-Tn5 transposase in the absence of Mg ++ , 

allowing the stable Transposome complex 

to form. 


The Transposome was then electroporated 

into the widely-used protein expression strain 

BL21(DE3). As described above, intracellular 

Mg ++ then activates the transposase, allowing 

DNA insertion to occur. Chromosomal 

insertions were selected for by plating on 

kanamycin-containing LB solid medium, and 

confirmed by screening for the lack of a 

circular plasmid, potentially due to uncut 

pMOD vector background. The presence of 

each insertion on the chromosome can be 

confirmed by purifying total cellular DNA 

using the MasterPure DNA Purification Kit 

(EPICENTRE) and performing sequencing 

reactions using primers that anneal to the ends 

of the inserted EZ-Tn5 transposon. 

Two different proteins were expressed in this 

manner to demonstrate the effectiveness of 

this approach. Three random chromosomal 

insertion clones were selected for each protein 

and assayed for the level of protein production 

as compared with the same proteins cloned 

into a pET T7 expression plasmid (Novagen). 

Protein yields were measured by lysing 

equivalent amounts of total cell mass and 

running them on an SDS-PAGE gel. The 

band intensities of the induced protein of 

interest were visually compared. 

As seen in FIG 2, the expression of Protein X 

when using a chromosomally inserted EZ-Tn5 

transposon was only very slightly lower than 

the expression achieved when using a pET 

plasmid. As for Protein Y, expression levels 

were very similar in E. coli cells, whether 

the protein was expressed using an EZ-Tn5 

transposon or from a pET plasmid (FIG 3). 

Conclusion 

Chromosomal protein expression using 

transposon-mediated insertion is a viable 

alternative for high-level expression of proteins 

in E. coli. EZ-Tn5 transposon-based expression 

resulted in production of similar amounts


www.EpiBio.com/pmod.asp 

FIG 2. Expression of protein 

X from a pET plasmid or a 

chromosomally-inserted EZ-Tn5 

transposon. Equivalent amounts of 

un-induced(-) and induced(+) total 

cell lysates based on OD 600 

values 

were separated on a 10% SDS- 

PAGE. Lanes 1-2, pET expressed 

protein X; Lane 3, 250ng purified 

protein X standard; Lanes 4-9, 

chromosomal EZ-Tn5 transposon 

protein X expression clones. 

EZ-Tn5 pMOD-2 Transposon 

Construction Vector 

MOD0602 20 µg 

EZ-Tn5 pMOD-3 

Transposon Construction Vector 

MOD1503 20 µg 

EZ-Tn5 pMOD-4 Transposon 

Construction Vector 

MOD4804 20 µg 

EZ-Tn5 pMOD-5 


MOD4805 20 µg 

NEW! EZ-Tn5 pMOD-6 


MOD7906 20 µg 

FIG 3: Expression of protein 

Y from a pET plasmid or a 

chromosomally-inserted EZ-Tn5 

transposon. Equivalent amount of 

un-induced(-) and induced(+) total 

cell lysates based on OD 600 

values 

were separated on a 10% SDS- 

PAGE. Lanes 1-2, pET expressed 

protein Y; Lane 3, 250ng purified 

protein Y standard; Lanes 4-9, 

chromosomal EZ-Tn5 transposon 

protein Y expression clones. 

www.EpiBio.com/transposase.asp 

EZ-Tn5 Transposase 

TNP92110 10 U 

www.EpiBio.com/ec100d_pir.asp 

TransforMax EC100D pir + 


ECP09500 5 x 100 µl 

Includes control vector containing an R6Kγori. 

of two different proteins as a commonly 

used plasmid expression system. EZ-Tn5 

Transposomes provide researchers with an 

easy-to-use, well-characterized tool for creating 

these chromosomal expression strains. 

Stable chromosomal insertion of heterologous 

genes in EZ-Tn5 transposons enable expression 

of high levels of proteins without use of 

antibiotics, which is desirable in all production 

situations, but especially when operating under 

pharmaceutical GMP conditions. The ability 

to stably maintain chromosomal expression 

strains without antibiotic selection additionally 

facilitates validation and verification of GMP 

production strains. Chromosomal expression 

of transposon-inserted genes also helps 

eliminate other problematic issues associated 

with plasmid based expression systems such 

as plasmid loss, and accumulation of excessive 

cellular debris from non-productive cells. 

Thus, chromosomal protein expression using 

the EZ-Tn5 system can increase recombinant 

protein yields, lower the costs associated 

with culture media, and simplify the protein 

purification process. 

In addition to its uses for manufacturing 

proteins, the EZ-Tn5 Transposome expression 

technology can be used to genetically engineer 

bacterial strains to express non-native proteins 

for specific tasks, such as bio-remediation 

or for conversion of biomass into useful 

materials, and for production of biofuels. 

EPICENTRE’s EZ-Tn5 Transposome 

expression technology is a powerful tool for 

researchers and bioengineers who wish to 

“customize” microbes or express proteins at 

high level for downstream applications. 

References 

1. Studier, F. W. and Moffatt, B. A. (1986) J. Mol. 

Biol. 189(1), 113. 

2. Friehs, K. (2004) Adv. Biochem. Eng. Biotechnol. 

86, 47. 

3. Jahic, M. et al., (2003) Microb. Cell Fact. 2(1), 6. 

4. Goryshin, I. W. et al., (2000) Nat. Biotechnol. 

18(1), 97. 

TransforMax EC100D pir-116 


EC6P095H 5 x 100 µl 

Includes control vector containing an R6Kγori. 

www.EpiBio.com/r6kgori_kan2_transposome.asp 

EZ-Tn5 Tnp 

Transposome Kit 

TSM08KR 10 Reactions 

www.EpiBio.com/kan2_tnp_transposome.asp 

EZ-Tn5 Tnp Transposome Kit 

TSM99K2 10 Reactions 

EZ-Tn5 Tnp Transposome Kit 

TSM99D1 10 Reactions 

Each kit contains a ready-to-use Transposome 

complex with either a kanamycin resistance marker 

or dihydrofolate reductase gene (for selection with 

trimethoprim) and forward and reverse primers for 

sequencing. 

This technology is covered by patents exclusively licensed to 

EPICENTRE. Nonprofit organizations receive a research license 

by purchase of EZ-Tn5 or HyperMu transposon tools (visit 

http://www.EpiBio.com/terms.asp#patents for detailed licensing 

information). For-profit organizations are allowed to use in vivo 

transposition technology and tools solely for evaluation purposes 

for up to three months, after which a license will be required for 

the continued use of the patented technology, tools and derivatives. 

For questions and inquiries please contact Stefan Koehler at 

stefan.koehler@EpiBio.com. 



21


Isolate Intron Lariat Loops with RNase R 

Les M. Hoffman and James Murray, EPICENTRE Biotechnologies 

Precursor mRNA or pre-mRNA is an 

incompletely processed single-strand of 

mRNA synthesized from a DNA template 

in the nucleus of a cell. The processing of 

pre-mRNA by splicing has long been of interest 

to researchers studying gene regulation. RNA 

splicing is a spliceosome mediated event that 

removes non-coding intervening sequences 

(introns) from pre-mRNA and releases them 

in the form of branched lariat structures 

that are later degraded. Alternative splicing 

locations of introns can have a dramatic 

effect on the resulting translation product. 

EPICENTRE’s Ribonuclease R (RNase R) 

can be used to study such splicing events by 

digesting mRNA from this complex mixture 

leaving the intron lariat loops intact (FIG 1). 

Ribonuclease R from E. coli, is a magnesiumdependent 

3′→5′ exoribonuclease that digests 

essentially all linear RNAs but not lariat or 

circular RNA structures.1,2 It also digests 

double-stranded RNA with 3′-overhangs 

shorter than seven nucleotides.2 Most cellular 

RNAs will be digested completely by RNase 

R, with the exception of tRNAs, 5S RNA, 

and intron lariats. The 3′-tails of lariats will 

be trimmed by RNase R to the branch 

point nucleotide, where there is a 2′,5′- 

phosphodiester linkage. Lariats produced 

during pre-mRNA splicing of intron regions 

can be isolated from a mixture of total RNA 

by digestion with RNase R. EPICENTRE’s 

ArrayPure Nano-scale RNA Purification 

Kit, MasterPure RNA Purification Kit, and 

MasterPure Yeast RNA Purification Kits 

are ideal products for obtaining total RNA 

preparations. 

RNA isolated using RNase R can be employed 

as a template to produce labeled cDNA with 

random primers. Intronic cDNA molecules can 

be used as a target for microarrays containing 

Pre-mRNA 

mRNA 

Ribonuclease R (RNase R) 

Exon 1 Intron Exon 2 

Intron 

Exon 1 Exon 2 

Exon 1 Exon 2 

Intron 

Intron 

RNase R Digestion 

Lariat Only Remains 

FIG 1. Introns are spliced out from pre-mRNA and 

released in the form of a branched lariat structure. 

RNase R degrades linear RNAs while preserving the loop 

portion of a lariat RNA. 

potential intron sequences, or for tiling arrays 

containing overlapping regions of complete 

chromosomes or genomes. The cDNA 

produced will not be a linear representation of 

the intron, as the lariat has no starting point 

of origin, but the sequences contained in it 

will be intron-derived. RNase R is provided 

in a 250 U size (20 U/µl or 1 µg/µl) and is 

supplied with a 10X Reaction Buffer. 

Applications 

• Alternative mRNA splicing studies. 

• Gene expression studies. 

• Intron cDNA production. 

• Intronic screening of cDNA libraries. 

i2220706 

References 

1. Suzuki, H. et al., (2006) Nucleic Acids Res. 34(8), 

e63. 

2. Vincent, H. A. and Deutscher, M.P.(2006) J. Biol. 

Chem. 281(40), 29769. 

www.EpiBio.com/rnaser.asp 

NEW! Ribonuclease R (RNase R) 

RNR07250 

250 Units 

www.EpiBio.com/arraypure.asp 

ArrayPure Nano-scale RNA Purification Kit 

MPS04050 

50 Purifications 

www.EpiBio.com/masterpure_complete.asp 

MasterPure RNA Purification Kit 

(for isolating RNA only) 

MCR85102 100 Purifications 

Contents: Red Cell Lysis Solution, Tissue and Cell 

Lysis Solution, MPC Protein Precipitation Reagent, 

2X T & C Lysis Solution, TE Buffer, RNase-Free 

DNase I, Proteinase K, and 1X DNase Buffer. 

www.EpiBio.com/masterpure_yrna.asp 

MasterPure Yeast RNA Purification Kit 

MPY03010 10 Reactions 

MPY03100 100 Reactions 

Contents: Extraction Reagent for RNA, MPC 

Protein Precipitation Reagent, TE Buffer (in 100 

rxn kit only), Proteinase K, RNase-Free DNase I, 

10X DNase Buffer, and 2X T & C Lysis Solution. 

Kits and Reagents for 

In Vitro Transcription 

& RNA Research 

Kits and Reagents for 

Nucleic Acid Extraction 

& Purification Research 

Ask for the latest literature available—EPICENTRE SPOTLIGHT Brochures 

22 




Talktidbits 

TransforMax EC100 E. Coli Competent Cells 

High Efficiency Electrocompetent & Chemically Competent E. Coli 

The highly versatile TransforMax 

EC100 E. coli competent cells are ideal 

for most cloning applications. The cells 

provide very high transformation efficiency 

when tested against a wide range of 

different sized supercoiled DNAs as well 

as DNA directly from a ligation reaction 

(see Table 1). 

Benefits 

• High transformation efficiency with 

clones of all sizes - including BAC clones 

(Table 1). 

• Lac ZΔM15 for blue/white screening of 

recombinants. 

• Restriction minus (mcrA, Δ(mrr-hsdRMSmcrBC)) 

enables efficient cloning of 

methylated DNA. 

• Endonuclease minus (endA1) to ensure 

high yields of DNA. 

• Recombination minus (recA1) for greater 

stability of large cloned inserts. 

Genotype 

F - mcrA Δ(mrr-hsdRMS-mcrBC) 

φ80dlacZΔM15 ΔlacX74 recA1 endA1 

araD139 Δ(ara, leu)7697 galU galK λ- rpsL 

nupG 

TransforMax EC100 E. coli Transformation Efficiencies with Different DNAs 

DNA 

TransforMax EC100 


• Transformation efficiency of >1 x 10 10 

cfu/µg of pUC19. 



EC10005 5 X 100 µl 

EC10010 10 X 100 µl 

Includes pUC19 control DNA. 


Chemically Competent E. coli 



pUC19 2.5 X 10 8 1.4 X 10 10 

8.1 kb Clone 1.3 X 10 7 Not tested 

13.1 kb Clone 4.3 X 10 6 1.3 X 10 9 

23.1 kb Clone 9.2 X 10 5 3.0 X 10 8 

145 kb BAC Clone Not tested 7 X 10 7 

13.1 kb Clone directly 

from a ligation reaction 

2.2 X 10 5 2.1 X 10 7 

Table 1. Comparison of the 

transformation efficiencies 

of TransforMax EC100 

E. coli with a variety of 

DNAs. Transformations were 

performed using 50 µl of 

competent cells and either 

supercoiled DNAs of the 

indicated sizes or a 1 µl aliquot 

from a standard 

10 µl ligation reaction. 

Results shown are in cfu/µg 

of DNA and are the average 

transformation efficiencies 

obtained from several trials. 

TransforMax EC100 Chemically 

Competent E. coli 

• Transformation efficiency of >1 x 10 8 

cfu/µg of pUC19. 

• Supplied in convenient single-use 50 µl 

aliquots. 

TransforMax EC100 Chemically 

Competent E. coli 

CC02810 10 X 50 µl 

Includes pUC19 control DNA. 

Phage T1-resistant TransforMax EC100 

Competent cells are also available. 

Please visit www.EpiBio.com for details. 


DNA Ligations in 5 Minutes! 

5 min. 

15 min. 

30 min. 

1 hr. 

2 hr. 

EPICENTRE’s Fast-Link DNA Ligation Kit 

is formulated to provide fast and efficient 

DNA ligations in as little as 5 minutes at 

room temperature for both routine and 

high-throughput cloning. 

FIG 1. Time curve for cohesive-end ligation using Fast- 

Link Kit. Lambda Hind III markers were ligated using 

2 units of Fast-Link DNA Ligase (Lanes 3–7). Lane 1, kb 

ladder; Lane 2, no enzyme. 

Fast and Efficient DNA Ligations 

• Ligate cohesive-end DNA in 5 minutes 

at room temperature. 

• Ligate blunt-end DNA in 15 minutes at 

room temperature. 

• Ligation of PCR product with A-overhangs 

in 1 hour or less at 16°C. 

• Transform cells without desalting the 

ligation. 

1 2 3 4 5 6 7 


LK11025 25 Ligations 

LK0750H 50 Ligations 

LK6201H 100 Ligations 



23

The new TargetAmp Nano-g Biotin-aRNA Labeling Kit for the Illumina ® System produces 

Biotin-aRNA (also called cRNA) with high signal intensity for use with the Illumina ® Gene 

Expression System, such as the Illumina ® Expression BeadChips. 

TargetAmp Nano-g Biotin-aRNA Labeling Kit for the Illumina ® System— 

• Detects more genes using less input total RNA than another kit. 

• Easy, 6-hour linear RNA amplification and labeling process 

preserves the relative transcript abundance of the sample. 

• Optimized for producing Biotin-aRNA with high signal intensity 

from as little as 25 ng of input total RNA. 

See Page 9 for more details—Or visit www.EpiBio.com 

and enter code: TNAX1 

1656 7896 268 

Input RNA 

100 ng 

Input RNA 

400 ng 

• EPICENTRE • Another 

TargetAmp Nano-g Supplier’s Kit 

Biotin-aRNA Labeling Kit 

for the Ilumina ® System 

Transcripts detected using Biotin-aRNA (also 

called cRNA) produced by a TargetAmp 

Nano-g Biotin-aRNA Labeling Kit for the 

Illumina ® System. The kit detects more genes 

using less input total RNA than Biotin-aRNA 

produced by another kit. 

i2260807a 

NEW Products! 

QuickExtract DNA Extraction Solution 

TAQXpedite PCR Systems 

Now also 

available for 

plants 

Get FAST 

PCR or qPCR 

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See Pages 13 & 14 for more details— 

Or visit www.EpiBio.com 

and enter code: QEAX6 

See Pages 15 & 16 for more details—Or visit www.EpiBio.com 

and enter code: TGAX4 

P-FORUM14:2

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