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