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.