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Pfu DNA Polymerase, Native, 2.5 U/Μl 100 U 500 U 20 Mm Tris-Hcl (Ph 8.8 at 25°C), 2 Mm Mgso4, 3

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Pfu DNA Polymerase, Native, 2.5 U/Μl 100 U 500 U 20 Mm Tris-Hcl (Ph 8.8 at 25°C), 2 Mm Mgso4, 3 DESCRIPTION PROTOCOL GUIDELINES FOR PREVENTING Thermo Scientific ™Pfu DNA Polymerase is a highly To prepare several parallel reactions and to minimize the CONTAMINATION OF PCR REACTION thermostable DNA polymerase from the possibility of pipetting errors, prepare a PCR master mix During PCR more than 10 million copies of template DNA hyperthermophilic archaeum Pyrococcus furiosus. The by mixing water, buffer, dNTPs, primers and template are generated. Therefore, care must be taken to avoid PRODUCT INFORMATION enzyme catalyzes the template-dependent polymerization DNA. Pfu DNA Polymerase should be the last component contamination with other templates and amplicons that Pfu DNA Polymerase, of nucleotides into duplex DNA in the 5’3’ direction. added. Prepare sufficient master mix for the number of may be present in the laboratory environment. General native Pfu DNA Polymerase also exhibits 3’5’ exonuclease reactions plus one extra to allow for pipeting error. recommendations to lower the risk of contamination are (proofreading) activity, which enables the polymerase to 1. Gently vortex and briefly centrifuge all solutions after as follows: Pub. No. MAN0012888 correct nucleotide incorporation errors. It has no 5’3’ thawing. Prepare your DNA sample, set up the PCR mixture, Rev. Date 3 June 2016 (B.00) exonuclease activity and no detectable reverse 2. Place a thin-walled PCR tube on ice and add the perform thermal cycling and analyze PCR products in transcriptase activity. separate areas. following components for each 50 µL reaction: The error rate of Pfu DNA Polymerase in PCR is 2.6x10-6 Set up PCR mixtures in a laminar flow cabinet Concentration: 2.5 U/µL errors per nt per cycle, as determined by the modified Water, nuclease-free (#R0581) variable equipped with an UV lamp. method described in (1). Wear fresh gloves for DNA purification and reaction Lot: __ Expiry Date: __ 10X Pfu Buffer with MgSO4* 5 µL Note. dUTP, dITP and primers containing these set up. nucleotides should not be used in PCR with Pfu DNA dNTP Mix, 2 mM each (#R0241) 5 µL (0.2 mM of each) Use reagent containers, dedicated for PCR. Use Store at -20°C Polymerase because the binding of this enzyme to DNA Forward primer 0.1-1.0 µM positive displacement pipettes, or pipette tips with templates with uracil and hypoxanthine stalls DNA aerosol filters to prepare DNA samples and perform synthesis (2,3). Reverse primer 0.1-1.0 µM PCR set up. Applications Use PCR-certified reagents, including high quality www.thermofisher.com Template DNA 50 pg - 1 µg High fidelity PCR. water (e.g., Water, nuclease-free, #R0581). Generation of PCR products for cloning and Pfu DNA Polymerase 1.25-2.5 U For Research Use Only. Not for use in diagnostic procedures. Always perform “no template control” (NTC) reactions expression. to check for contamination. RT-PCR for cDNA cloning and expression. Total volume 50 µL Carryover contamination control in conjunction with Generation of PCR product for blunt-end cloning (4). *If using 10X Pfu Buffer without MgSO4, a 25 mM MgSO4 UDG is not applicable using Pfu DNA Polymerase. Site-directed mutagenesis. solution should be added to 50 µL of the master mix: Source Final Pyrococcus furiosus concentration 1 1.25 1.5 1.75 2 2.5 3 4 Ordering Information Definition of Activity Unit of MgSO4, mM One unit of the enzyme catalyzes the incorporation of GUIDELINES FOR PRIMER DESIGN Pfu DNA polymerase (native) 10 nmol of deoxyribonucleotides into a polynucleotide Volume of Use the Thermo Scientific REviewer primer design 2 2.5 3 3.5 4 5 6 8 #EP0571 #EP0572 fraction in 30 min at 72°C. 25 mM MgSO4, µL software at www.thermofisher.com /reviewer or follow Component 100 U 500 U Enzyme activity is assayed in the following mixture: general recommendations for PCR primer design as Pfu DNA polymerase, native, 2.5 U/µL 100 U 500 U 20 mM Tris-HCl (pH 8.8 at 25°C), 2 mM MgSO4, 3. Gently vortex the samples and spin down. outlined below: 10 mM (NH4)2SO4, 10 mM KCl, 0.1 mg/mL BSA, PCR primers are generally 20-30 nucleotides long. 10X Pfu Buffer with MgSO4 0.6 mL 2x1.25 mL 4. If using a thermal cycler that does not use a heated lid, 0.1% (v/v) Triton X-100, 0.75 mM activated salmon milt overlay the reaction mixture with 25 µL of mineral oil. Optimal GC content of the primer is 40-60%. Ideally, 10X Pfu Buffer 0.6 mL 2x1.25 mL DNA, 0.2 mM of each dNTP, 0.4 MBq/mL [3H]-dTTP. 5. Perform PCR using the following thermal cycling C and G nucleotides should be distributed uniformly 25 mM MgSO4 0.6 mL 2x1.25 mL Storage Buffer conditions: along the primer. The enzyme is supplied in: 20 mM Tris-HCl (pH 8.2), Avoid placing more than three G or C nucleotides at Temperature, Number of 1 mM DTT, 0.1 mM EDTA, 100 mM KCl, Step Time the 3’-end to lower the risk of non-specific priming. 0.1% (v/v) Nonidet P40, 0.1% (v/v) Tween 20 and °C cycles If possible, the primer should terminate with a G or C 50% (v/v) glycerol. Initial denaturation 95 1-3 min 1 at the 3’-end. 10X Pfu Buffer with 20 mM MgSO4 Avoid self-complementary primer regions, Denaturation 95 30 s 200 mM Tris-HCl (pH 8.8 at 25°C), 100 mM (NH4)2SO4, complementarities between the primers and direct 100 mM KCl, 1 mg/mL BSA, 1% (v/v) Triton X-100, Annealing Tm-5 30 s 25-35 primer repeats to prevent hairpin formation and primer 20 mM MgSO4. dimerization. Extension 72 2 min/kb 10X Pfu Buffer 200 mM Tris-HCl (pH 8.8 at 25°C), 100 mM (NH4)2SO4, Final extension 72 5-15 min 1 100 mM KCl, 1% (v/v) Triton X-100, 1 mg/mL BSA. Inhibition and Inactivation Inactivated by gel purification or phenol/chloroform (continued on reverse page) extraction. The 3’5’ exonuclease activity associated with Pfu dNTPs Number of cycles DNA Polymerase may degrade the primers. It is The recommended final concentration of each dNTP is The number of cycles may vary depending on the amount therefore important that Pfu DNA Polymerase be 0.2 mM. In certain PCR applications, higher dNTP of template DNA in the PCR mixture and the expected added last to the reaction mixture. Use the longer concentrations may be necessary. Due to the binding of PCR product yield. 2+ primers (20-30 bp) with higher CG content. Mg to dNTPs, the MgSO4 concentration needs to be In most cases, 25-35 cycles are sufficient. adjusted accordingly. It is essential to have equal Check for possible sites of undesired complementarity Final extension between primers and template DNA. concentrations of all four nucleotides (dATP, dCTP, dGTP and dTTP) present in the reaction mixture. After the last cycle, it is recommended to incubate the When designing degenerate primers, place at least PCR mixture at 72°C for additional 5-15 min to fill-in any 3 conservated nucleotides at the 3’-end. To achieve 0.2 mM concentration of each dNTP in the PCR mixture, use the following volumes of dNTP mixes: possible incomplete reaction products. Differences in melting temperatures (Tm) between the dNTP Mix, dNTP Mix, dNTP Mix, Troubleshooting two primers should not exceed 5°C. Volume of 2 mM each 10 mM each 25 mM each For troubleshooting please visit www.thermofisher.com PCR mixture Estimation of primer melting temperature (#R0241) (#R0191) (#R1121) For primers containing less than 25 nucleotides, the CERTIFICATE OF ANALYSIS approx. melting temperature (Tm) can be calculated 50 µL 5 µL 1 µL 0.4 µL 25 µL 2.5 µL 0.5 µL 0.2 µL Endodeoxyribonuclease Assay using the following equation: Tm = 4 (G + C) + 2 (A + T), 20 µL 2 µL 0.4 µL 0.16 µL No detectable degradation was observed after incubation where G, C, A, T represent the number of respective of supercoiled plasmid DNA with Pfu DNA polymerase. nucleotides in the primer. Primers Exodeoxyribonuclease Assay If the primer contains more than 25 nucleotides The recommended concentration range of the PCR specialized computer programs e.g., REviewer™ primers is 0.1-1 µM. Excessive primer concentrations No detectable degradation was observed after incubation increase the probability of mispriming and generation of of single stranded and double stranded radiolabeled (www.thermofisher.com /reviewer) are recommended to account for interactions of adjacent bases, effect of salt non-specific PCR products. For degenerate primers and oligonucleotides with enzyme. primers used for long PCR higher primer concentrations concentration, etc. Functional Assay in the range of 0.3-1 µM are often favorable. Pfu DNA Polymerase was tested for amplification of COMPONENTS OF THE REACTION MIXTURE 950 bp single copy gene from human genomic DNA. CYCLING PARAMETERS Template DNA Quality authorized by: Jurgita Zilinskiene Initial DNA denaturation Optimal amounts of template DNA in the 50 µL reaction volume are 0.05-1 ng for both plasmid and phage DNA, It is essential to completely denature the template DNA at and 0.1-1 µg for genomic DNA. Higher amounts of the beginning of PCR to ensure efficient utilization of the LIMITED USE LABEL LICENSE: Internal Research and Development template increase the risk of generating of non-specific template during the first amplification cycle. If the GC References Use Only. PCR products. Lower amounts of template reduce the content of the template is 50% or less, an initial 1-3 min 1.
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