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  • Protocol for a PCR Reaction using Phusion Hot Start DNA Polymerase

    Overview

    Carefully mix and centrifuge all tubes before opening to improve recovery. When using Phusion Hot Start DNA Polymerase, it is not necessary to perform the PCR setup on ice. Prepare a master mix for the appropriate number of samples to be amplified. The DNA polymerase should be pipetted carefully and gently as the high glycerol content (50%) in the storage buffer may otherwise lead to pipetting errors.

    Protocol

    1. Prepare the following in a 0.5 ml eppendorf tube:
    2. Gently mix the reaction and spin in a centrifuge.
      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 PCR reaction: