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  • FAQ: How should I set up an amplification reaction using Taq DNA Polymerase?

    We recommend assembling all reaction components on ice and quickly transferring the reactions to a thermocycler preheated to the denaturation temperature (95°C).

    COMPONENT
    25 μl
    REACTION
    50 μl
    REACTION
    FINAL
    CONCENTRATION
    10X Reaction Buffer
    2.5 μl
    5 μl
    1X
    10 mM dNTPs
    0.5 μl
    1 μl
    200 μM
    10 μM Forward Primer
    0.5 μl
    1 μl
    0.2 μM (0.05-1 μM)
    10 μM Reverse Primer
    0.5 μl
    1 μl
    0.2 μM (0.05-1 μM)
    Taq DNA Polymerase
    0.125 μl
    0.25 μl
    1.25 units/50 μl PCR
    Template DNA variable
    variable
    <1,000 ng
    Nuclease-Free Water to 25 μl to 50 μl
     
    Notes: Gently mix the reaction.  Collect all liquid to the bottom of the tube by a quick spin if necessary.  Overlay the sample with mineral oil if using a PCR machine without a heated lid.

    Transfer PCR tubes from ice to a PCR machine with the block preheated to 95°C and begin thermocycling:
    STEP
    TEMP
    TIME
    Initial Denaturation 95°C
    30 seconds
    30 Cycles 95°C
    45-68°C
    68°C
    15-30 seconds
    15-60 seconds
    1 minute/kb
    Final Extension 68°C
    5 minutes
    Hold 4-10°C