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 |
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 |
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.