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.
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