Fig.1. Restriction map of vector ptz57r/t. EcoRI 615 Ecl136II 621 SacI 621 Acc65I 627 KpnI 627 Bsp68I 633 Mva1269I 637 Mph1103I 639 XbaI 644.

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Dwain Horn
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1 1 2 InsTAclone PCR Cloning Kit (#K1214, for 30 reactions) (Kit for direct cloning of the PCR products with the TransformAid Bacterial Transformation System) Fermentas InsTAclone PCR Cloning Kit is a convenient system for direct one-step cloning of PCR-amplifi ed DNA fragments. DNA polymerases, that lacking 3' 5' exonuclease activity ( proofreading, see Table 1), possess deoxynucleotidyl transferase (TdT) activity in addition to primer extension activity, which frequently results in the addition of extra adenines at 3'-ends of amplifi ed DNA molecules [1]. This becomes an annoying problem when such fragments are to be cloned into conventional plasmid vectors as blunt-ended. Additional treatment of the 3'-ends, such as end polishing of the amplifi ed DNA fragment with Klenow fragment [2], T4 DNA polymerase [3], or E.coli Exonuclease III [4] is usually necessary. It should be noted that the 3'-end extension activity of Taq DNA polymerase is nucleotide-specifi c with respect to the last 3'-end nucleotide [6]. For example, the enzyme adds an extra dg or da nucleotide if the 3'-end nucleotide of the PCR fragment is dg, and makes a 3'-dA overhang if the 3 -end nucleotide is dc (see Table 2). Fermentas offers a fast and effi cient one-step InsTAclone PCR Cloning Kit which allows the cloning of Taq-amplifi ed PCR fragments without any post-pcr treatment. The TA cloning method is especially suitable for cloning of PCR fragments amplifi ed with primers that carry dg or dc at their 5 -ends. The high effi ciency of the kit is based on the use of a specifi cally designed cloning vector, ptz57r/t (see Fig.1). The vector has been pre-cleaved with Eco32I (an isoschizomer of EcoRV) and treated with terminal deoxynucleotidyl transferase to create 3 - overhangs at both ends. When a PCR fragment with 3 -da overhangs is ligated into the Fig.1. Restriction map of vector ptz57r/t. EcoRI 615 Ecl136II 621 SacI 621 Acc65I 627 KpnI 627 Bsp68I 633 Mva1269I 637 Mph1103I 639 XbaI 644 BamHI 654 Cfr9I 658 Eco88I 658 SmaI 658 ApaI 661 Bsp120I 661 HincII 667 SalI 667 XmiI 667 PstI 672 AlfI 674 Eco147I 678 PaeI 684 HindIII 690 T7 promoter 1 2

2 3 4 5' vector 3' 5' vector 3' Table 1. Exonucleolytic activities of thermostable DNA polymerases and structure of PCR product ends. 5' 3' da 3' 5' P-dA DNA fragment vector, a circular molecule with two nicks (see Fig.2) is produced. The circular product can be used directly to transform E.coli cells with high effi ciency. An additional advantage of this approach is that the T-overhangs prevent recircularization of the vector during the ligation procedure. As a result, the yields of the recombinants are typically as high as 90%. The DNA insert can be readily excised from the versatile polylinker of ptz57r/t and subcloned into other vectors, as well as sequenced using standard M13/pUC primers. da 5' da-p 5' 3' Fig. 2. Ligation of a PCR fragment into the ptz57r/t vector. 3' DNA polymerase 5' 3' exonuclease activity 3' 5' exonuclease activity PCR products ends Taq 1 yes no 3'-A overhangs Tth 2 yes no 3'-A overhangs Tfl 2 yes no 3'-A overhangs Stoffel Fragment 3 no no 3'-A overhangs Vent R (exo ) 4 no no 70% blunt, 30% single base overhangs 5 Tli, Vent R no yes >95% blunt 5 Deep Vent R no yes >95% blunt Pfu 6 no yes blunt Pwo 7 no yes blunt Compiled from: 1 - Clark, J.M., Nucl. Acids Res. 16, , Life Science Catalog, Promega, PCR Systems, Reagents and Consumables, Perkin Elmer, /01 Catalog, New England Biolabs, Kong, H., Kucera, R.B., Jack, W.E., J. Biol. Chem. 268, , Catalog, Stratagene, Biochemicals Catalog, Roche Molecular Biochemicals,

3 5 6 Table 2. Structure of the 3'-ends of PCR products generated by Taq DNA polymerase [6]. 5'-end nucleotide of primer A C G T 3'-end nucleotide of product on complementary strand T T, +A G +G>+A>+C C +A>+C A (+A) at a low effi ciency + - addition of extra nucleotide (3'-overhang) - deletion of the complementary nucleotide (3'-recession) PCR products generated by DNA polymerases that possess 3' 5' exonuclease ( proof-reading ) activity (see Table 1) have > 95% blunt ends, and after phosphorylation of 5'-end they can be ligated by vector with blunt ends. In order to maintain the high effi ciency of the InsTAclone PCR Cloning Kit, competent cells with a transformation effi ciency of at least 10 6 per µg of supercoiled DNA should be used. The TransformAid Bacterial Transformation System is included in this kit for the preparation of competent cells that have consistently high transformation effi ciency. COMPONENTS OF THE KIT 1. Vector ptz57r/t: 90µl (4.95µg) of plasmid vector ptz57r/t, 0.055µg/µl. 2. 5X Ligation Buffer: 0.3ml of ready-to-use ligation buffer. 3. T4 DNA Ligase, 5u*/µl: 30µl of the enzyme solution in storage buffer: 20mM Tris-HCl, ph 7.5 (25 C), 1mM DTT, 50mM KCl, 0.1mM EDTA and 50% glycerol. 4. Control PCR Fragment: 20µl (0.84µg) of 953 bp control PCR fragment, for 5 control ligation reactions, 0.042µg/µl. 5. Control DNA (ptz57r with insert): 30µl of 3838 bp supercoiled control plasmid vector ptz57r DNA with insert, for 10 control electrophoreses, 0.1µg/µl. 6. Vector ptz57r DNA (without insert): 30µl of 2886 bp supercoiled plasmid vector ptz57r DNA, for 10 control electrophoreses, 0.1µg/µl. 7. Water, nuclease-free: 1.5ml of 0.22µm membrane-fi ltered molecular biology grade water. 8. TransformAid Bacterial Transformation System**: a) 70ml (2 x 35ml) of C-Medium. b) 5ml (4 x 1.25ml) of T-Solution (A). c) 5ml (4 x 1.25ml) of T-Solution (B). * One unit of enzyme catalyzes the conversion of 1nmol of [ 32 PPi] into Norit -adsorbable form in 20 minutes at 37 C (Weiss unit). ** Components of Transformation System do not contain glucose. 5 6

4 7 8 7 STORAGE All components should be stored at -20 C. The C-Medium should be stored at -20 C (in case of long-term storage) or at 4 C. It would be more convenient to keep the working C-Medium at 4 C since thawing is not required prior to use. PROTOCOL 1. Ligation 2. Transformation 3. Analysis of Recombinant Clones 1. Ligation The effi ciency of ligation is known to depend greatly on the purity of PCR fragments. If the PCR product is suffi ciently clean (a homogeneous band of desired size is observed on the gel), it can be directly used in the ligation reaction. If the PCR template contains the β-lactamase (ampicillin resistance) gene, DNA purifi cation is necessary. Due to ability of such template molecules to confer antibiotic resistance, a high level of background colonies may appear. The use of unpurifi ed PCR products (if additional bands or primerdimers are present) usually leads to a decreased yield of the target clones and a higher number of background constructs. Following PCR amplifi cation, DNA fragments suitable for ligation can be prepared by purifi cation in agarose gels [8], by ultrafi ltration [9] or chromatography [9-11]. For recovery of DNA fragments from agarose gels we recommend Fermentas DNA Extraction Kit (#K0513). Extraction with organic solvents [8], centrifugation through microcolumns [15] or digestion of agarose with agarase (#EO0461) are usually suffi cient. The effi ciency of ligation also depends on the amount of added nucleotides to the 3 -ends of the PCR products. We recommend a fi nal long extension step be included in your PCR protocol. A 20-30min extension step at C will result in an increased amount of the PCR product with the extra nucleotides added. This usually results in a 3-4 fold higher yield of recombinant colonies. Another factor affecting the effi ciency of ligation and subsequent clone selection is the vector/insert ratio in the ligation reaction; the optimal molar ratio of the ends appears to be around 1:3, respectively [8]. The amount of DNA insert required for effi cient ligation with 0.15µg (0.18pmol ends) of the vector can be approximately determined from Table 3. Table 3. Conversion table for the amount of a PCR fragment required per ligation reaction. Length of DNA fragment (bp) 8 pmoles of ends per 1µg of DNA Quantity of PCR fragments for ligation reaction in µg (0.54pmol ends)

5 9 10! Dissolve the purifi ed PCR fragment in 10-20µl of TE buffer, determine the approximate DNA concentration by agarose gel electrophoresis and visual comparison to a known amount of DNA size markers (#SM0383, #SM0393, #SM0403, #SM0241, #SM0331, #SM1113). Determine the amount of PCR fragment equivalent to 0.54pmoles of ends by referring to Table 3. " Add the following components into a 1.5ml microcentrifuge tube: vector ptz57r/t, (0.165µg, 0.18pmol ends) 3µl, purifi ed PCR fragment, (approx. 0.54pmol ends) 4µl, 5X Ligation Buffer 6µl, Water, nuclease-free, up to 29µl, T4 DNA Ligase, 5u 1µl. # Incubate at 22 C for 1 hour. For maximum yield of useful recombinants, the reaction time can be extended (to overnight). Note A control ligation reaction can be performed using 4µl (168ng, 0.54pmol ends) of Control PCR Fragment provided with the kit. 2. Transformation using the TransformAid Bacterial Transformation System Competent cells of the following E.coli strains have been tested for use with the kit: JM107, JM109, XL1-Blue, SURE, NM522 (see Fermentas Catalog - Bacterial Strain Genotypes). The following mutations are important for transformation: - enda1 (mutation in the DNA-specifi c endonuclease I gene), it helps to increase the yield and quality of plasmids; - reca1 (mutation in general recombination gene); - lacz M15 (partial deletion of β-galactosidase gene), this mutation is required for the blue/white selection; - laci q (mutation leads to high levels of the lac repressor protein, inhibiting background transcription from the lac promoter). The TransformAid Bacterial Transformation System is included for the transformation step. The TransformAid system provides a consistent and effi cient method for obtaining competent cells and transformation Preparation of Bacteria from Overnight Bacterial Culture! Inoculate 2ml of TransformAid C-Medium with bacteria from frozen stock or a colony. Incubate the culture overnight at 37 C in a shaker. " Pre-warm culture tubes containing the required amount of TransformAid C-Medium (1.5ml for each of 2 transformations) at 37 C. # Add 1/10 volume of the overnight culture to the pre-warmed C-Medium (0.15ml overnight culture for each 1.5ml C-Medium). Incubate the tubes in a shaker at 37 C for 20min. Note A maximum of 26 transformations can be performed from 2ml of overnight culture. The culture can be kept at 4 C for at least a week and used for preparation of fresh culture. 9 10

6 Preparation of Bacteria from Bacterial Culture Plate! Inoculate a LB plate with bacteria using the streak plate method, and incubate the plate overnight at 37 C. " Pre-warm culture tubes containing the required amount of TransformAid C-Medium (1.5ml for each of 2 transformations) at 37 C. # Move a small portion of bacterial culture (4x4mm size for each 1.5ml of C-Medium) from the overnight LB plate using an inoculating loop into the pre-warmed C-Medium. Suspend the culture by gently mixing and incubate the tubes in a shaker at 37 C for 2 hours. Note The colonies on LB plates can be stored at 4 C and used for inoculating fresh cultures within 10 days Transformation Procedure! Pre-warm LB-Ampicillin agar plates (see Note 1) in a 37 C incubator for at least 20min. " Prepare TransformAid T-Solution by mixing equal volumes of T-Solution (A) and T-Solution (B) (500µl of solution for each of 2 transformations). Keep TransformAid T-Solution on ice (see Note 2). # Dispense 1.5ml of fresh culture into a microcentrifuge tube and spin at maximum speed for 1min at room temperature (RT) or at 4 C (see Note 3). % Discard the supernatant and resuspend the pelleted cells in 300µl of TransformAid T-Solution. Incubate the tubes on ice for 5min. 11 & Spin down the cells again for 1min at RT or at 4 C and then remove the supernatant. ' Resuspend the cells in 120µl of TransformAid T-Solution, and incubate on ice for 5min. ( Prepare DNA for transformation by dispensing 1µl of supercoiled DNA (10-100pg) or 2.5µl of ligation mixture (10-20ng of vector DNA) into new microcentrifuge tubes and sit them on ice for 2min (see Note 4). ) Add 50µl of the resuspended cells to each tube containing DNA and incubate them on ice for 5min. * Plate the cells on pre-warmed LB-Ampicillin agar plates. Incubate the plates overnight at 37 C. For control transformation use 2µl (10ng vector DNA) of the ligation reaction mixture with Control PCR DNA fragment (see Note on p.9). Incubation overnight at 37 C usually results in colonies per plate (about 90% of which are white). Note 1. DNA containing other antibiotic resistance genes can be used in the TransformAid system. The transformation effi ciency should fi rst be tested with supercoiled DNA containing these antibiotic genes. In our hands, DNA containing tetracycline and chloramphenicol resistance genes have similar transformation effi ciency to DNA containing the ampicillin resistance gene (~10 6 to 10 7 ). However, DNA containing the kanamycin resistance gene has a lower transformation effi ciency (~10 5 ). 2. After thawing T-Solution (A) and T-Solution (B), mix contents thoroughly prior to combining equal volumes of T-Solution (A) and T-Solution (B). 12

7 The centrifugation can be carried out at room temperature (RT), but the cells should be kept on ice at all other times as indicated. Do not leave the cells in the centrifuge at RT for more than 1min as this will signifi cantly decrease the transformation effi ciency. 4. The quantity of DNA used in transformation will infl uence the transformation effi ciency. The transformation effi ciency usually decreases with an excess of DNA. In the TransformAid system, approximately 100 to 2000 colonies will be obtained using 100pg of supercoiled DNA for most bacterial strains (JM107, JM109, XL1-Blue, SURE, NM522). To transform cells with the ligation reaction mixture, a reaction volume not larger than 2.5µl (containing 10-20ng of vector DNA) should be used. We usually obtain colonies for ligation reactions Scale-up When preparing a large volume of competent cells for transformation, the transformation procedure can be scaled up proportionately. The cells can be pelleted in large centrifuge tubes at ,000 x g for 5min in steps 3 and Preparation of LB-Ampicillin Plates! Prepare LB Medium (per liter): Bacto Tryptone 10g Bacto Yeast extract 5g NaCl 10g " Dissolve solids in 800 ml of water, adjust ph to 7.5 with NaOH. # Adjust water to a fi nal volume of 1000ml. % Autoclave the medium. Prepare LB-Ampicillin plates: & Add 15g agar to 1 liter of LB medium. ' Microwave the medium until the agar dissolves. ( Allow the solution to cool down to 55 C, then add ampicillin to a fi nal concentration of 50µg/ml. (For convenience, prepare a stock solution of ampicillin at 25mg/ml. Use 2ml of the stock solution per liter of LB-agar medium. Store the stock solution at -20 C.) ) Mix gently, and pour 30-35ml of LB-agar medium directly onto each plate. IPTG (isopropyl-β-d-thiogalactopyranoside) ( #R0391, #R0392) stock solution (0.1M): 1.2g IPTG, add water to 50ml fi nal volume. Filter-sterilize and store at 4 C. Use 40µl per plate. X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) (#R0401, #R0402) stock solution: 20mg/ml in N,N-dimethylformamide. Use 40µl per plate. Note The concentration of antibiotic in the agar plates will affect the number of colonies obtained. The antibiotic resistance of transformants is dependent on the copy number of the plasmid used. When a low copy number plasmid is used, the concentration of the antibiotic can be decreased in order to obtain a greater number of transformants

8 Analysis of Recombinant Clones Analyse 4-6 white colonies for the presence and orientation of the DNA insert using one of the following methods: Restriction analysis. Isolate plasmid DNA from an overnight bacterial culture using a convenient plasmid miniprep method. To speed up the process and to assure the quality of purifi ed plasmid DNA, use the GeneJET Plasmid Miniprep Kit (#K0501). To digest DNA from recombinant clones in just 5 minutes, use FastDigest restriction enzymes. Colony PCR. Use the M13/pUC Sequencing Primers (#SO100, #SO101, #SO114, #SO115). Sequencing. Use the M13/pUC Sequencing Primers (#SO100, #SO101, #SO114, #SO115). TROUBLESHOOTING Problem Possible Cause Remedy Few or no transformants Poor quality of the competent cells Unsuccessful ligation The use of DNA polymerases with proof-reading activity Perform a test transforma tion using known amount of supercoiled plasmid DNA (>10 6 colonies per µg of supercoiled DNA is expected) Perform a control ligation reaction using Control PCR DNA fragment (see p.9) Ligate the insert to a bluntend vector or perform PCR with Taq DNA polymerase High background of nonrecombinants Unsuccessful PCR fragment purifi cation Repurify DNA fragment (see p.7) QUALITY CONTROL All components of the kit are tested using the control PCR fragment. Yield of target clones is >90%. Quality authorized by: Gintaras Gulbinas Low antibiotic concentration Ratio of vector ptz57r/t DNA to PCR fragment is too high E.coli strain contains intact reca1 and enda1 genes Supply LB medium with fresh antibiotic (see p.14) Adjust ratio to optimal (see p.9) Choose a E.coli strain with reca1 and enda1 mutations for transformations (see p.10) 15 16

9 17 18 References 1. Clark, J.M., Nucl. Acids Res., 16, , Williams, A.W., Amplifi cations, 3, 19, Stoker, A.W., Nucl. Acids Res., 18, 4290, Kaluz, S. et al., Nucl. Acids Res., 20, , Marchuk, D. et al., Nucl. Acids Res., 19, 1154, Hu,G., DNA Cell Biol., 12, , Weiss, B. et al., J. Biol. Chem., 243, , Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY, Krowczynska, A.M. et al., BioTechniques, 13, , Katz, E. et al., BioTechniques, 8, , Warren, W. et al., BioTechniques, 10, , Vogelstein, B. and Gillespie, D., Proc. Natl. Acad. Sci. USA, 76, 615, Poloquin, J.J. et al., BioTechniques, 10, , Errington, J., Nucl. Acids Res., 18, 3254, Schwarz, H. et al., BioTechniques, 13, , Thuring, R.W. et al., Anal. Biochem., 66, , Hanahan, D., J. Mol. Biol., 166, , Hanahan, D., Techniques for Transformation of E.coli. DNA Cloning. Vol1/Ed.D.M.Glover-Oxford, Washington DC:IRLPress, , Alexander, D.C. et al., Gene, 1, 79-89, Michelsen, B.K., Analytical Biochemistry, 225, , Birnboim, H.C. and Doly, J., Nucl. Acids Res., 7, , Del Sal, G., Manfi oletti, G. and Schneider, C., Nucl. Acids Res., 16, 9878, Morelle, G., Focus,11, 7-8, Sounders, S. E. and Burke, J.F., Nucl. Acids Res., 18, 4948, Wang, L.M. et al., BioTechniques, 6, , Chowdbury, K., Nucl. Acids Res., 19, 2792, Clewell, D.B. and Helinski, D.B., Proc. Natl. Acad. Sci. USA, 62, , Attal, J.C., Puissant, L. and Hondebine, L.-M., BioTechniques, 8, , Griffi th, D.M., BioTechniques, 6, , Raymond, G.J., Bryant III, K., Nelson, A. and Johnson, J.D., Anal. Biochem., 173, , Marko, M.A., Chipperfi eld, R. and Birnboim, H.C., Anal. Biochem., 121, , Ishaq, M., Wolf, B. and Ritter, C., BioTechniques, 9, 19-24,

10 19 20 Trademarks Bacto Tryptone and Bacto Yeast Extract are the registered trademarks of Difco Laboratories GmbH. InsTAclone is a trademark of Fermentas. Norit is a registered trademark of American Norit Company. SURE is a registered trademark of Stratagene Cloning Systems. TransformAid is a trademark of Fermentas. ElutaTube is a trademark of Fermentas. (3) Revised

Rapid DNA Ligation & Transformation Kit

1 Rapid DNA Ligation & Transformation Kit (#K1432 for 30 reactions) INTRODUCTION The Rapid DNA Ligation & Transformation Kit enables ligation of any type of DNA in 5 minutes at ambient temperature followed