Protocols and Applications Guide (US Letter Size) - Promega

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||||||||||||| 13Cloning I. Introduction The cloning of genes, gene fragments and other DNA sequences is a fundamental part of molecular biology. To study the function of a particular DNA sequence, you must be able to manipulate that sequence. There are two main ways to achieve this: the polymerase chain reaction (PCR) and the more traditional use of restriction enzymes and modifying enzymes to “cut and paste” the desired DNA fragments into cloning vectors, which can then be replicated using live cells, most commonly E. coli. The use of PCR has an advantage in that it gives you the option to re-amplify the target DNA each time your DNA supplies dwindle without ligation into a vector or transformation into E. coli. Alternatively, PCR products can be ligated into a suitable vector, which can then be transformed into and replicated by E. coli. This chapter covers the basics of cloning using PCR and restriction enzymes, including DNA cleanup prior to ligation, ligation, transformation and screening to identify recombinant clones. The PCR process is a useful tool to quickly and easily amplify the desired sequences. With the successful sequencing of whole and partial genomes of organisms across all biological kingdoms, DNA cloning by PCR is an easily attainable option. Public DNA databases allow researchers to design primers to amplify their DNA fragment of interest directly from the genomic DNA of the desired organism. With the simple addition of a reverse transcription step prior to PCR, RNA sequences can be converted to cDNA, which can then be cloned into a suitable vector. For additional information about amplification of DNA and RNA sequences using PCR, see the Protocols and Applications Guide chapter on PCR Applications (www.promega.com/paguide/chap1.htm). PCR products generated using a nonproofreading DNA polymerase such as Taq DNA polymerase, which lacks 3´→5´ exonuclease activity, have a single templateindependent nucleotide at the 3´ end of the DNA strand (Clark, 1988; Newton and Graham, 1994). This single-nucleotide overhang, which is most commonly an A residue, allows hybridization with and cloning into T vectors, which have a complementary 3´ single T overhang. PCR products generated using a proofreading DNA polymerase, such as Pfu DNA polymerase, have blunt ends and must be cloned into a blunt-ended vector or need a single 3´A overhang added to ligate into a T vector (Knoche and Kephart, 1999). If PCR amplification of the desired DNA fragment is not possible or desirable, restriction enzyme digestion of the target DNA can be employed. The desired fragment may need to be separated from other DNA fragments in the reaction, so the size of the desired DNA fragment should be known. Once isolated, the fragment is cloned into a vector with compatible ends. If the vector ends are capable of religating (e.g., the vector has blunt ends or is cut with a single restriction enzyme), the vector is often treated with alkaline phosphatase to discourage recircularization and maximize ligation between the insert and vector. Protocols & Applications Guide www.promega.com rev. 3/09 Following transformation into E. coli, the resulting bacterial colonies are screened by PCR for the correct recombinant vector using primers to amplify the insert. Alternatively, the recombinant vector can be identified by performing a restriction enzyme digestion to determine the presence of the correct insert. Screening is often simplified by using vectors that contain an antibiotic-resistance gene, so cells containing the vector will survive on medium supplemented with the appropriate antibiotic. Screening can be further simplified by choosing a vector and E. coli strain that are compatible with blue/white screening, which takes advantage of intracistronic α-complementation to regenerate β-galactosidase activity. Many E. coli strains used for cloning and propagation of plasmids contain a chromosomal deletion of the lac operon but carry an F´ episome that provides the remaining coding sequence of the lacZ gene. The functional lacZ gene product, β-galactosidase, is produced when the lacZ coding information missing on the F´ episome is provided by the information contained in the plasmid. This activity is detected by plating bacteria transformed by plasmids on plates containing isopropyl β-D-thiogalactopyranoside (IPTG; an inducer of the lac promoter) and 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-Gal; a dye that produces a blue color when hydrolyzed by β-galactosidase). When the reading frame of the α peptide is disrupted by insertion of a foreign DNA fragment or deletion of vector sequences, α-complementation does not occur, and the bacterial colonies remain white or occasionally light blue. II. Promega Products for Cloning A. Thermostable DNA Polymerases The use of amplification enzymes is the first step in cloning by PCR. Most people use PCR for cloning, taking advantage of the single nucleotide A overhang left after amplification with a nonproofreading DNA polymerase to ligate the amplimer to a vector containing T overhangs. However, products will be blunt-ended if the DNA polymerase has 3′→5′ exonuclease activity, also known as proofreading activity. Alternatively, PCR primers can add sequences for restriction enzyme sites, and these resulting products can be digested and ligated into a vector with compatible ends. Promega provides several thermostable DNA polymerases. These include the GoTaq® Amplification Family, Tfl DNA polymerase and proofreading polymerases. A detailed listing of the various enzymes for use in PCR can be found in the Protocol and Applications Guide chapter on PCR Applications (www.promega.com/paguide/chap1.htm), in the section "Thermostable DNA Polymerases". The GoTaq® Amplification Family of products is highlighted in the following section. GoTaq® Amplification Family GoTaq® DNA Polymerase is available in various formulations to suit your needs: the standard GoTaq® DNA Polymerase, which is supplied with 1.5mM MgCl2 in the 1X reaction buffer; GoTaq® Flexi DNA Polymerase, which PROTOCOLS & APPLICATIONS GUIDE 13-1
||||||||||||| 13Cloning allows a range of MgCl2 to be added for PCR; and GoTaq® Green Master Mix, which is a premixed, ready-to-use solution containing GoTaq® DNA Polymerase, dNTPs, MgCl2 and reaction buffers at optimal concentrations for efficient amplification of DNA templates by PCR. All GoTaq® products contain Taq DNA polymerase in a proprietary formulation that offer enhanced amplification over conventional Taq DNA polymerase. Each member of the GoTaq® family has a reaction buffer that contains two dyes (a blue dye and a yellow dye) that separate during electrophoresis to show migration progress as well as a compound that increases sample density. Samples can be loaded directly onto gels without the need to add a separate loading dye. If the dyes interfere with your downstream applications, GoTaq® DNA Polymerases are supplied with a 5X Colorless Reaction Buffer. Alternatively, the PCR Master Mix offers a ready-to-use formulation without any dyes. Reaction products generated with these systems contain A overhangs and are ready for T-vector cloning. Additional Resources for GoTaq® DNA Polymerase Promega Publications PN083 Introducing GoTaq® DNA Polymerase: Improved amplification with a choice of buffers (www.promega.com/pnotes/83/) Citations Theodoropoulos, G. et al. (2006) Determination of prevalence and risk factors of infection with Babesia in small ruminants from Greece by polymerase chain reaction amplification. Vet. Parasitol. 135, 99–104. Researchers used GoTaq® DNA Polymerase to test sheep and goat blood samples for the presence of Babesia DNA. Primers were designed around the 18S rRNA sequence of Babesia sp. PCR was performed in a 50µl reaction volume using 1 unit of GoTaq® DNA Polymerase. Ten microliters of each amplification reaction were loaded on gels and subjected to electrophoresis. PubMed Number: 16139956 Weinberg, J.B. et al. (2005) Acute respiratory infection with mouse adenovirus type 1. Virology 340, 245–54. Mouse adenovirus type 1 (MAV-1) was detected in DNA extracted from the lungs of mice by PCR amplification of the E1A region of MAV-1. For these assays, 80ng of total DNA was added to a 20µl PCR reaction containing 0.5 units of GoTaq® DNA Polymerase, 4µl of 5X GoTaq® Buffer, dNTPs and primers for MAV-1 E1A. The amplified products were separated on a 1.8% agarose gel and stained with ethidium bromide. PubMed Number: 16054189 Protocols & Applications Guide www.promega.com rev. 3/09 Additional Resources for GoTaq® Flexi DNA Polymerase Promega Publications PN089 GoTaq® Flexi DNA Polymerase: Robust performance with magnesium optimization (www.promega.com/pnotes/89/) Additional Resources for GoTaq® Green Master Mix Promega Publications eNotes Activity of Promega Restriction Enzymes in GoTaq® Green Master Mix and PCR Master Mix (www.promega.com /enotes/applications/ap0075.htm) eNotes Analyses of gene disruption by whole-cell PCR using the GoTaq® Green Master Mix (www.promega.com /enotes/applications/ap0065.htm) PN091 GoTaq® Green Master Mix: From amplification to analysis (www.promega.com/pnotes/91/) PN101 Recombinant clone screening using the GoTaq® Hot Start Green Master Mix (www.promega.com /pnotes/101/17252_13/17252_13.html) Citations Rendón, M.A. et al. (2007) Commensal and pathogenic Escherichia coli use a common pilus adherence factor for epithelial cell colonization. Proc. Natl. Acad. Sci. USA. 104, 10637–42. The authors identified an adherence factor of enterohemorrhagic E. coli that is involved in colonization of cultured epithelial cells. This factor, named E. coli common pilus (ECP), is encoded by the ecpA gene, which is present 96% of E. coli strains tested, as determined by PCR. The remaining 4% of the strains were deficient in the ECP operon, as determined by multiplex PCR amplification of ecpR, ecpA, ecpB and ecpC sequences. PCR were performed using GoTaq® Green Master Mix. An ecpA deletion mutant exhibited impaired adherence compared to the wildtype E. coli strain. Complementation of the mutant strain with the plasmid pMR13, the pGEM®-T Vector containing the ecpA gene, restored the strain's ability to adhere to epithelial cells. PubMed Number: 17563352 Song, J.H. et al. (2006) Human astrocytes are resistant to Fas ligand and tumor necrosis factor-related apoptosisinducing ligand-induced apoptosis. J. Neurosci. 26, 3299–308. Total RNA was extracted from human astrocytes and control A549 cells. First strand cDNA was synthesized from 3µg of total RNA using random hexamers. PCR was performed on the cDNA samples using primers for DR4, DR5 and GAPDH with GoTaq® Green Master Mix. The PROTOCOLS & APPLICATIONS GUIDE 13-2
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CONTENTS I. Nucleic Acid Amplificat
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| 1Nucleic Acid Amplification CONTE
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| 1Nucleic Acid Amplification Unamp
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| 1Nucleic Acid Amplification Capoz
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| 1Nucleic Acid Amplification is co
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| 1Nucleic Acid Amplification One i
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| 1Nucleic Acid Amplification React
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| 1Nucleic Acid Amplification comig
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| 1Nucleic Acid Amplification inclu
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| 1Nucleic Acid Amplification polym
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| 1Nucleic Acid Amplification Addit
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| 1Nucleic Acid Amplification 3. Pl
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| 1Nucleic Acid Amplification 13. S
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| 1Nucleic Acid Amplification IX. R
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| 1Nucleic Acid Amplification Hoppe
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|| 2RNA Interference CONTENTS I. RN
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|| 2RNA Interference zebrafish (War
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|| 2RNA Interference Additional Res
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|| 2RNA Interference 2. Combine sep
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|| 2RNA Interference Target Site Se
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|| 2RNA Interference Transient Tran
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|| 2RNA Interference VII. Reference
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|| 2RNA Interference Wianny, F. and
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||| 3Apoptosis I. Introduction A. C
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|||| 4Cell Viability CONTENTS I. In
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||||| 5Protein Expression I. Introd
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||||||| 7Cell Signaling CONTENTS I.
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Table 8.1. pGL4 Luciferase Reporter
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||||||||| 9DNA Purification I. Intr
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|||||||||| 10Cell Imaging I. Introd
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