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

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
    Deep 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, Deep Vent DNA Polymerase can be diluted in  Diluent D (NEB #B8004S) or 1X reaction buffer. For example, 1 µl of Deep Vent 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 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