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  • FAQ: How should I set up a PCR using Multiplex PCR 5X Master Mix?

    Assemble reactions on ice.  A manual hot start practice is recommended to improve specificity.  Add the master mix last and start PCR cycling immediately.  Add the following components to a thin-walled PCR tube on ice:
     COMPONENT 25 μl
    REACTION
     50 μl
    REACTION
    FINAL
    CONCENTRATION
    Multiplex PCR 5X
    Master Mix
     5 μl
     10 μl
     1X
    1 μM Primer Stock
     3.75 μl
    7.5 μl
     0.15 μM
    (0.05-0.4 μM)
    Template DNA
     variable  variable  <1,000 ng
     Nuclease-Free Water
     to 25 μl
    to 50 μl
     
    Note: 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 to a PCR machine with the block preheated to 95°C and begin the programmed cycling:
     STEP TEMP
    TIME
     Initial Denaturation
    95°C
    1 minute
     30-40 Cycles
    95°C
    55-68°C
    68°C
    20 seconds
    1 minute
    1-2 minutes/kb*
     Final Extension
     68°C  5 minutes
     Hold 4-10°C
     
    *The specified extension time is based on the longest amplicon in the reaction.  For example, in a 4-plex reaction of 200, 300, 400 and 500 base pairs we recommend an extension time of 30 seconds to 1 minute at 68°C.

    Usage Notes:
    • Use high quality primers (desalted or HPLC purified).
    • Accurately quantify and adjust primer stock concentrations to 50 μM in 0.5X TE Buffer.
    • Individually test the PCR primer pairs, preferably in a temperature-gradient PCR machine.
    • Mix all primers equally at 1 μM in 0.5X TE buffer.
    • Test multiplex PCR with equal molar concentration of all primer pairs, preferably in a temperature-gradient PCR machine.