2.1.3. PCR conditions
PCR bias is known to occur in multiple template PCR amplifications, leading to a situation where the relative concentrations of templates are not equal to the relative concentrations of products. To minimize PCR bias we recommend using no more than 25 PCR cycles, performing reactions in triplicate, and then combining triplicate reactions before sequencing.
- Use HotStarTaq Plus Master
Mix Kit (QIAGEN, Valencia, CA) for 50 μl reaction.
a. 25 μl HotStarTaq plus master mix (2x).
b. 2 μl each of 10 μM forward and reverse primer (0.4 μM final concentration).
c. 5 μl DNA template (100 ng).
d. 16 μl sterilized nanopure water.
- PCR conditions.
a. 95°C for 5 minutes.
b. 25 cycles of: 94°C for 30 seconds, 60°C for 40 seconds, 72°C for 1 minute.
c. Final elongation of 72°C for 10 minutes.
- PCR purification
a. Remove unincorporated primers, dNTPs, etc., using a PCR purification kit, such as Agencourt AMPure XP (Agencourt Bioscience Corporation, MA) or MinElute PCR columns (QIAGEN, Valencia, CA).