Home Protocols Guidelines for PCR Optimization for Vent DNA Polymerase

Guidelines for PCR Optimization for Vent DNA Polymerase

Introduction

When using Vent, Deep Vent, Vent (exo-) or Deep Vent (exo-) DNA Polymerases the basic reaction conditions are 1X ThermoPol® Reaction Buffer, DNA template, DNA polymerase, 1-6 mM MgSO4, 200-400 µM each dNTP and 0.1-0.5 µM each primer. The three most important variables to optimize are the amount of polymerase, the annealing temperature for the primer and the magnesium level. Each new primer:template may require reoptimization. This protocol highlights guidelines for optimizing your reaction.

Buffers

ThermoPol Reaction Buffer Pack (NEB# B9004)
ThermoPol II (Mg-free) Reaction Buffer Pack (NEB# B9005)
ThermoPol DF (Detergent-free) Reaction Buffer Pack (NEB# B9013)

Protocol

  1. Enzyme Amount: It is important to use the optimal amount of enzyme, especially with the proofreading DNA polymerases. Start with 1 unit/100 µl reaction volume for proofreading DNA polymerases or 4 units/100 µl reaction volume for exo- derivatives (for different reaction volumes adjust this ratio accordingly). In general, lower DNA template concentrations in a primer extension reaction necessitate using the lower amount of DNA polymerase within the recommended range. Recommended ranges are 1.0-2.0 units per 100 µl reaction volume for the Vent, and Deep Vent DNA Polymerases, and 2-4 units for the Vent (exo-) and Deep Vent (exo-) DNA Polymerases.
  2. Annealing Temperature: The optimal annealing temperature for the primer can usually be predicted from any of several standard methods of calculation. If this temperature does not give satisfactory results, the annealing temperature should be examined in 3°C increments. We recommend using NEB's Tm Calculator to determine appropriate annealing temperatures for PCR.
  3. Magnesium Concentration: The optimal magnesium concentration is usually 2-6 mM. If EDTA is present at significant levels in DNA added to your reaction, the test range may need to be extended higher. For Vent and Deep Vent DNA Polymerases, primer extensions longer than 2 kb almost always require magnesium levels higher than 2 mM, while for primer extensions shorter than 2 kb, there is no correlation between length and optimum magnesium concentration.