Protocol for Taq 5X Master Mix PCR Reaction


These recommendations serve as a starting point; in order to maximize amplification the reaction conditions may require optimization (see "Taq 5X Master Mix Guidelines for PCR Optimization" protocol).


  1. Thaw Taq 5X Master Mix at room temperature and mix well by inverting several times before use.
  2. Prepare the following reaction in a thin-walled PCR tube on ice:
    Component 25 µl reaction 50 µl reaction Final Conc.
    Taq master Mix (5X) 5 µl 10 µl 1X
    Upstream Primer (10 μM stock) 0.5 µl 1 µl 0.2 µM
    Downstream Primer (10 μM stock) 0.5 µl 1 µl 0.2 µM
    Template DNA determined bu user determined by user <500 ng
    Nuclease-free water to 25 µl to 50 µl  
  3. 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.
  4. Cycling conditions for a routine PCR reaction:
    Initial Denaturation    94-95°C    1 minute
    25-40 Cycles 94-95°C
    20 seconds
    30 seconds
    1 minute per kb
    Final Extension 68°C 5 minutes
    Final Soak 4-10°C