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  • Protocol for a Routine Deep 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 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 (exo-) 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 (exo-) DNA Polymerase can be diluted in Diluent D (NEB #B8004S) or 1X reaction buffer. For example, 1 µl of Deep 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. 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