Home Protocols Protocol for a Routine Vent PCR

Protocol for a Routine Vent 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 Vent DNA Polymerase Guidelines for PCR Optimization Protocol).

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 DNA Polymerase* 0.25-0.5 μl 0.5-1 unit
    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 DNA Polymerase can be diluted just prior to the reaction in 1X reaction buffer. For example, 1 μl of Vent 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 PCR:

    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