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.