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Standard PCR (reaction set up)
This protocol is for the Standard PCR (reaction set up)
The provided set-up is suitable for following Thermo Scientific enzymes: DreamTaq™ DNA Polymerase, DreamTaq™ Green DNA Polymerase, Taq DNA Polymerase, recombinant and Taq DNA Polymerase, native. Master Mix. To prepare several parallel reactions and to minimize the possibility of pipetting errors, prepare a PCR master mix by adding water, buffer, dNTPs, primers and Taq DNA polymerase. Prepare enough master mix for the number of reactions and add one extra to compensate for pipetting errors. Aliquot the master mix into individual PCR tubes and add template DNA. 1. Gently vortex and briefly centrifuge all solutions after thawing. 2. Place a thin-walled PCR tube on ice and add the following components for each 50 µl reaction: 10X Taq buffer dNTP Mix, 2 mM each Forward primer Reverse primer 25 mM MgCl2* Template DNA Taq DNA Polymerase Water, nuclease-free Total volume 5 µl 5 µl (0.2 mM of each) 0.1-1 µM 0.1-1 µM 1-4 mM 10 pg – 1 µg 1.25 u to 50 µl 50 µl 3. * O ptional for PCR with DreamTaq™ DNA Polymerase, because the provided 10X DreamTaq™ buffer contains 20 mM MgCl2. 4. Gently vortex the samples and spin down to collect drops. Note • When using thermal cyclers without a heated lid, overlay the reaction mixture with 25 µl of mineral oil. • Reaction volumes can be scaled up or down as long as the final concentrations of the reaction components remain the same. Recommended thermal cycling conditions: Step Initial Denaturation Denaturation Annealing Extension Final Extension Temperature, °C 95 95 Tm-5 72 72 Time, min 1-3 0.5 0.5 1 min/kb 5-15 1 25-40 Number of Cycles 1
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