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  • Protocol for a Routine Taq PCR

    Introduction

    All components should be mixed and spun down prior to pipetting. These recommendations serve as a starting point; in order to maximize amplification the reaction conditions may require optimization (see Taq DNA Polymerase Guidelines for PCR Optimization protocol).

    Protocol

    1. Prepare the following 50 µl reaction in a 0.5 ml PCR tube on ice:

      * Due to the difficulties in pipetting small volumes of enzyme, Taq DNA Polymerase can be diluted in Diluent F (NEB #B8006S ) or 1X reaction buffer. For example, 1 µl of Taq DNA Polymerase is mixed with 4 µl of diluent and 1 µl of that mixture is added to the reaction. Enzyme diluted in Diluent F can be stored at -20°C for future use.
      Component 25 μl reaction 50 μl reaction Final Concentration
      10X ThermoPol or Standard Taq Reaction Buffer 2.5 µl 5 μl 1X
      10 mM dNTPs 0.5 µl 1 μl 200 µM
      10 µM Forward Primer 0.5 µl 1 μl 0.2 µM (0.05–1 µM)
      10 µM Reverse Primer 0.5 µl 1 μl 0.2 µM (0.05–1 µM)
      Template DNA variable variable <1,000 ng
      Taq DNA Polymerase* 0.125 µl 0.25 µl 1.25 units/50 µl PCR
      Nuclease-free water to 25 µl to 50 µl  
    2. Gently mix the reaction and spin down in microcentrifuge.
      If the thermocycler does not have a heated cover, add one drop of mineral oil to the reaction tube to prevent evaporation.

    3. Cycling Conditions for a Routine PCR:

      STEP
      TEMP
      TIME
      Initial Denaturation
      95°C
      30 seconds
      30 Cycles 95°C
      45-68°C
      68°C
      15-30 seconds
      15-60 seconds
      1 minute/kb
      Final Extension 68°C
      5 minutes
      Hold 4-10°C