Quick Protocol for Multiplex PCR 5X Master Mix

Overview

Assemble reactions on ice. A manual hot-start practice is recommended to improve specificity. Add the master mix last and start the PCR cycling immediately.

Protocol

  1. Add the following components to a thin-walled PCR tube:
    Component 25 µl reaction 50 µl reaction Final Conc.
    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 <1000 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 with mineral oil if using a PCR machine without a heated lid.
  2. Transfer PCR tubes to a PCR machine with the block preheated to 95°C and begin the programmed cycling:
    Initial Denaturation    95°C    1 minute
    30-40 Cycles 95°C
    55-68°C
    68°C
    20 seconds
    1 miunte
    1-2 minute per 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 or 60 seconds at 68°C.