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

    We recommend assembling all reaction components on ice and quickly transferring the reactions to a thermocycler preheated to the denaturation temperature (94°C).

    COMPONENT
    25 μl
    REACTION
    50 μl
    REACTION
    FINAL
    CONCENTRATION
    5X OneTaq Reaction Buffer*
    5 μl
    10 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)
    OneTaq 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
     
    *OneTaq GC Reaction Buffer and High GC Enhancer can be used for difficult amplicons

    **For amplicons between 3–6 kb, use 2.5–5 units/50 µl rxn

    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 94°C and begin thermocycling:
    STEP
    TEMP
    TIME
    Initial Denaturation 94°C
    30 seconds
    30 Cycles 94°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