Description PROTOCOL GUIDELINES FOR PREVENTING Taq DNA is a highly thermostable DNA To prepare several parallel reactions and to minimize the CONTAMINATION OF PCR REACTION polymerase from the thermophilic bacterium Thermus possibility of pipetting errors, prepare a PCR master mix by During PCR more than 10 million copies of template DNA PRODUCT INFORMATION aquaticus . The catalyzes 5’→3’ synthesis of DNA, mixing water, buffer, dNTPs, primers and Taq DNA are generated. Therefore, care must be taken to avoid Taq DNA Polymerase has no detectable 3’→5’ () activity Polymerase. Prepare sufficient master mix for the number contamination with other templates and amplicons that may (native, with BSA) and possesses 5’→3’ exonuclease activity. In addition, Taq of reactions plus one extra. Aliquot the master mix into be present in the laboratory environment. General DNA Polymerase exhibits deoxynucleotidyl individual PCR tubes and then add template DNA. recommendations to lower the risk of contamination are as #EP0072 500 U activity, which frequently results in the addition of extra 1. Gently vortex and briefly centrifuge all solutions after follows: at the 3’end of PCR products. thawing. • Prepare your DNA sample, set up the PCR mixture, Native Taq DNA Polymerase is preferred for amplification of Concentration: 5 U/L perform thermal cycling and analyze PCR products in bacterial DNA sequences homologous to those found in 2. Place a thinwalled PCR tube on ice and add the following Lot: __ separate areas. E.coli . components for each 50 Lreaction: • Set up PCR mixtures in a laminar flow cabinet equipped Expiry Date: __ Taq DNA Polymerase (native, with BSA) supplied with BSA 10X Taq Buffer 5 L with an UV lamp. Store at 20°C as a stabilizing agent. This version of Taq DNA Polymerase dNTP Mix, 2 mM each (#R0241) 5 L (0.2 mM of each) is often the best choice when amplifying DNA samples of • Wear fresh gloves for DNA purification and reaction set Forward primer 0.1 1.0 M lower purity. up. Reverse primer 0.1 1.0 M • Use reagent containers dedicated for PCR. Use positive Applications displacement pipettes, or use pipette tips with aerosol 25 mM MgCl 2* 14 mM • Routine PCR amplification of DNA fragments up to 5 kb (1). filters to prepare DNA samples and perform PCR set up. Template DNA 10 pg 1 g • Generation of PCR product for TA cloning. • Use PCRcertified reagents, including high quality water BSA included • DNA labeling (24). Taq DNA Polymerase 1.25 U (e.g., Water, nucleasefree, (#R0581)). www.thermoscientific.com/onebio • DNA sequencing (5). Water, nuclease free (#R0581) to 50 L • Always perform “no template control” (NTC) reactions to check for contamination. Source Total volume 50 L YT1 cells. *Volumes of 25 mM MgCl 2, required for specific final MgCl 2 GUIDELINES FOR PRIMER DESIGN Definition of Activity Unit concentration: Use the Thermo Scientific REviewer™ primer design Final concentration of MgCl 2, mM 1 1.5 2 2.5 3 4 One unit of the enzyme catalyzes the incorporation of software at www.thermoscientific.com/reviewer or follow Volume of 25 mM MgCl 2 for 50 L 10 nmol of deoxyribonucleotides into a polynucleotide 2 3 4 5 6 8 general recommendations for PCR primer design as outlined reaction, L fraction (adsorbed on DE81) in 30 min at 70 °C. below: Enzyme activity is assayed in the following mixture: 3. Gently vortex and spin down the samples. • PCR primers are generally 1530 long. 67 mM TrisHCl (pH 8.8 at 25 °C), 6.7 mM MgCl 2, 4. When using a thermal cycler that does not use a heated • Optimal GC content of the primer is 4060%. Ideally, 1 mM 2mercaptoethanol, 50 mM NaCl, 0.1 mg/mL BSA, lid, overlay the reaction mixture with 25 L of mineral oil. C and G nucleotides should be distributed uniformly along 0.75 mM activated salmon milt DNA, 0.2 mM of each dNTP, 5. Perform PCR using recommended thermal cycling the primer. Ordering Information 0.4 MBq/mL [3H]dTTP. conditions: • Avoid placing more than three G or C nucleotides at the Taq DNA Polymerase (native) Storage Buffer Temperature, Number of 3’end to lower the risk of nonspecific priming. Step Time Component #EP0072 The enzyme is supplied in: 20 mM TrisHCl (pH 8.0), 1 mM DTT, °C cycles • If possible, the primer should terminate with a G or C ® at the 3’end. Taq DNA Polymerase, 5 U/ L 500 U 0.1 mM EDTA, 100 mM KCl, 0.5% (v/v) Nonidet P40, Init ial denaturation 95 13 min 1 0.5% (v/v) Tween ® 20 and 50% (v/v) glycerol. • Avoid selfcomplementary primer regions, 10X Taq Buffer with KCl 2 × 1.25 mL Denaturation 95 30 s complementarities between the primers and direct primer 10X Taq Buffer with KCl 10X Taq Buffer with (NH 4)2SO 4 2 × 1.25 mL repeats to prevent hairpin formation and primer Annealing Tm5 30 s 2540 100 mM TrisHCl (pH 8.8 at 25 °C), 500 mM KCl, dimerization. 25 mM MgCl 2 2 × 1.25 mL 0.8% (v/v) Nonidet P40. Extension 72 1 min/kb • Check for possible sites of undesired complementary 20 mg/ mL BSA 0.25 mL between primers and template DNA. 10X Taq Buffer with (NH 4)2SO 4 Final Extension 72 515 min 1 • When designing degenerate primers, place at least 750 mM TrisHCl (pH 8.8 at 25 °C), 200 mM (NH 4)2SO 4, 3 conservated nucleotides at the 3’end. Note 0.1% (v/v) Tween 20. • When introducing restriction enzyme sites into primers, refer • When amplifying DNA samples of lower purity, BSA may Inhibition and Inactivation to the table “Cleavage efficiency close to the termini of PCR help to avoid PCR inhibition. fragments” located on www.thermoscientific.com/onebio to • Inhibitors: ionic detergents (deoxycholate, sarkosyl • The recommended concentration of BSA in the reaction determine the number of extra bases required for efficient and SDS) at concentrations higher than 0.06, 0.02 and mixture is 0.1mg/mL. cleavage. 0.01%, respectively (6). • Differences in melting temperatures (Tm) between the two • Inactivated by phenol/chloroform extraction. primers should not exceed 5 °C. Rev.13

(continued on reverse page) Estimation of primer melting temperature To achieve 0.2 mM concentration of each dNTP in the PCR Extension CERTIFICATE OF ANALYSIS For primers containing less than 25 nucleotides, the approx. mixture, use the following volumes of dNTP mixes: The optimal extension temperature for Taq DNA Polymerase Endodeoxyribonuclease Assay melting temperature (Tm) can be calculated using the dNTP Mix, dNTP Mix, dNTP Mix, is 7075 °C. The recommended extension step is 1 min Volume of following equation: 2 mM each 10 mM each 25 mM each at 72 °C for PCR products up to 2 kb. For larger products, No conversion of covalently closed circular DNA to nicked PCR mixture Tm= 4 (G + C) + 2 (A + T), (#R0241) (#R0191) (#R1121) the extension time should be prolonged by 1 min/kb. DNA was detected after incubation of 10 U of Taq DNA Polymerase with 1 g of pUC19 DNA for 16 hours at 72 °C. where G, C, A, T represent the number of respective 50 L 5 L 1 L 0.4 L Number of cycles nucleotides in the primer. Exodeoxyribonuclease Assay 25 L 2.5 L 0.5 L 0.2 L The number of cycles may vary depending on the amount of If the primer contains more than 25 nucleotides 20 L 2 L 0.4 L 0.16 L template DNA in the PCR mixture and the expected PCR No degradation of DNA was observed after incubation of specialized computer programs e.g., REviewer product yield. 1 g of lambda DNA/HindIII fragments with 10 U Taq DNA (www.thermoscientific.com/reviewer ) are recommended to If less than 10 copies of the template are present in the Polymerase for 16 hours at 72 °C. account for interactions of adjacent bases, effect of salt Primers reaction, about 40 cycles are required. For higher template concentration, etc. Assay The recommended concentration range of the PCR primers amounts, 2535 cycles are sufficient. is 0.11 M. Excessive primer concentrations increase the No contaminating RNase activity was detected after COMPONENTS OF THE REACTION MIXTURE probability of mispriming and generation of nonspecific PCR Final extension incubation of 10 U of Taq DNA Polymerase with 1 g of products. After the last cycle, it is recommended to incubate the PCR [3H]RNA for 4 hours at 70 °C. Template DNA For degenerate primers higher primer concentrations in the mixture at 72°C for additional 515 min to fillin any possible Functional Assay Optimal amounts of template DNA in the 50 L reaction range of 0.31 M are often favorable. incomplete reaction products. If the PCR product will be volume are 0.011 ng for both and phage DNA, and cloned into TA vectors (for instance, using Thermo Scientific Taq DNA Polymerase was tested for amplification of 950 bp 0.11 g for genomic DNA. Higher amount of template CYCLING PARAMETERS InsTAclone PCR Cloning Kit (#K1213)), the final extension single copy gene from human genomic DNA and for increases the risk of generation of nonspecific PCR step may be prolonged to 30 min to ensure the highest amplification of cDNA. products. Lower amount of template reduces the accuracy of Initial DNA denaturation efficiency of 3’dA tailing of PCR product. If the PCR product Quality authorized by: Jurgita Zilinskiene the amplification. It is essential to completely denature the template DNA at will be used for cloning using Thermo Scientific CloneJET All routine DNA purification methods are suitable for the beginning of PCR to ensure efficient utilization of the PCR Cloning Kit (#K1231), the final extension step can be template preparation e.g., Thermo Scientific GeneJET™ template during the first amplification cycle. If the GC content omitted.

Genomic DNA Purification Kit (#K0721) or GeneJET Plasmid of the template is 50% or less, an initial 13 min denaturation

Miniprep Kit (#K0502). Trace amounts of certain agents used at 95°C is sufficient. For GCrich templates this step should Troubleshooting for DNA purification, such as phenol, EDTA and proteinase For troubleshooting please visit be prolonged up to 10 min. If longer initial denaturation step K, can inhibit DNA polymerase. Ethanol precipitation and is required, or the DNA is denatured at a higher temperature, www.thermoscientific.com/onebio repeated washes of the DNA pellet with 70% ethanol Taq DNA Polymerase should be added after the initial normally removes trace contaminants from DNA samples. denaturation step to avoid a decrease in its activity.

MgCl 2 concentration Denaturation 2+ NOTICE TO PURCHASER: Due to the binding of Mg to dNTPs, primers and DNA A DNA denaturation time of 30 seconds per cycle at 95 °C Use of this product is covered by US Patent No. 6,127,155. The purchase templates, Mg 2+ concentration needs to be optimized for is normally sufficient. For GCrich DNA templates, this step of this product includes a limited, nontransferable immunity from suit maximal PCR yield. The recommended concentration range can be prolonged to 34 min. DNA denaturation can also be under the foregoing patent claims for using only this amount of product for is 14 mM. If the Mg 2+ concentration is too low, the yield of enhanced by the addition of either 1015% glycerol or the purchaser’s own internal research. No right under any other patent PCR product could be reduced. On the contrary, nonspecific claim, no right to perform any patented method and no right to perform 10% DMSO, 5% formamide or 11.5 M betaine. The melting commercial services of any kind, including without limitation reporting the PCR products may appear and the PCR fidelity may be temperature of the primertemplate complex decreases results of purchaser's activities for a fee or other commercial reduced if the Mg 2+ concentration is too high. significantly in the presence of these reagents. Therefore, References consideration, is conveyed expressly, by implication, or by estoppel. This If the DNA samples contain EDTA or other metal chelators, the annealing temperature has to be adjusted accordingly. 1. Innis, M.A., et al., PCR Protocols and Applications: A Laboratory product is for research use only. Diagnostic uses under Roche patents the Mg 2+ concentration in the PCR mixture should be In addition, 10% DMSO and 5% formamide inhibit DNA Manual, Academic, New York, 1989. require a separate license from Roche. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied increased accordingly (1 molecule of EDTA binds one Mg 2+ ). 2. Celeda, D., et al., PCR amplification and simultaneous digoxigenin by 50%. Thus, the amount of the enzyme incorporation of long DNA probes for fluorescence in situ hybridization, Biosystems, 850 Lincoln Centre Drive, Foster City, California should be increased if these additives are used. dNTPs BioTechniques, 12, 98102, 1992. 3. Finckh, U., et al., Producing singlestranded DNA probes with the The recommended final concentration of each dNTP is Primer annealing Taq DNA polymerase: a high yield protocol, BioTechniques, 10, 3539, 0.2 mM. In certain PCR applications, higher dNTP The annealing temperature should be 5°C lower than the 1991. PRODUCT USE LIMITATION concentrations may be necessary. Due to the binding of melting temperature (Tm) of the primers. Annealing for 4. Yu, H. et al., Cyanine dye dUTP analogs for enzymatic labeling of This product is developed, designed and sold exclusively for research 2+ DNA probes, Nucleic Acids Res., 22, 32263232, 1994. purposes and in vitro use only. The product was not tested for use in Mg to dNTPs, the MgCl 2 concentration needs to be 30 seconds is normally sufficient. If nonspecific PCR diagnostics or for drug development, nor is it suitable for administration to adjusted accordingly. It is essential to have equal 5. Innis, M.A., et al., DNA sequencing with Thermus aquaticus DNA products appear, the annealing temperature should be polymerase and direct sequencing of polymerase chain reaction humans or animals. Please refer to www.thermoscientific.com/onebio for concentrations of all four nucleotides (dATP, dCTP, dGTP optimized stepwise in 12°C increments. When additives amplified DNA, Proc. Natl. Acad. Sci. USA, 85, 94369440, 1988. Material Safety Data Sheet of the product. and dTTP) present in the reaction mixture. which change the melting temperature of the primertemplate 6. Weyant, R.S., et al., Effect of ionic and nonionic detergents on the complex are used (glycerol, DMSO, formamide and betaine), Taq polymerase, Biotechniques, 9, 309308, 1990. © 2014 Thermo Fisher Scientific, Inc. All rights reserved. Tween is a registered trademark of ICI America, Inc. Nonidet is a trademark of Shell. 7. Lundberg, K.S., et al., Highfidelity amplification using a thermostable the annealing temperature must also be adjusted. All other trademarks are the property of Thermo Fisher Scientific Inc. and its DNA polymerase isolated from Pyrococcus furiosus, Gene, 108, 16, subsidiaries. 1991.