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.
- Add the following components to a thin-walled PCR tube:
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.
||25 µl reaction
||50 µl reaction
|Multiplex PCR 5X Master Mix
|1 μM Primer Stock
||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:
* 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.
1-2 minute per kb*