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  • Protocol for a Routine Vent (exo-) PCR

    Overview

    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 Guidelines for PCR Optimization for Vent DNA Polymerase).

    Buffers

    ThermoPol Reaction Buffer (NEB# B9004)
    Diluent D (NEB# B8004)

    Protocol

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

       COMPONENT VOLUME (μl)
      FINAL CONCENTRATION
       ThermoPol Reaction Buffer (10X)
      5 μl
      1X
       Deoxynucleotide (dNTP) Solution Mix (10 mM)
      1 μl
      200 μM
       Upstream Primer (10 μM stock)
      0.5-2.5 μl
      0.1-0.5 μM
       Downstream Primer (10 μM stock)
      0.5-2.5 μl
      0.1-0.5 μM
       DNA Template
      determined by user 
       Vent (exo-) DNA Polymerase*
      0.5-1.0 μl
      1-2 units
       MgSO4 (Optional)
      (1-6 mM)
      Nuclease-free water
      Bring reaction to a final volume of 50 μl

      * Due to the difficulties in pipetting small volumes of enzyme, Vent (exo-) DNA Polymerase can be diluted in Diluent D (NEB #B8004S) or 1X reaction buffer. For example, 1 µl of Vent (exo-) DNA Polymerase is mixed with 4 µl of diluent and 1 µl of that mixture is added to the reaction. Enzyme diluted in Diluent D can be stored at -20°C for future use.

    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 reaction:
      STEP TEMP TIME
      Initial Denaturation
      95°C
      2-5 minutes
      20-30 Cycles
      95°C
      55-65°C
      72°C
      15-30 seconds
      15-30 seconds
      1 minute per kb
      Final Extension
      72°C 5 minutes
      Hold 4-10°C