Home Protocols Protocol for a Routine Vent (exo-) PCR

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)

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 just prior to the reaction in 1X reaction buffer. For example, 1 μl of Vent (exo-) DNA Polymerase is mixed with 4 μl of 1X buffer and 1 μl of that mixture is added to the reaction.
  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