In order to improve the productivity of the genotyping processing, we think it is important to find the efficient master mixes for the PCR which leads to save our labors.

The points to pick up the right master mix we are looking at are

1. Pre-mixed and Ready to Use

2. Available on the first cycle program, within 1 hour in total

Finally we chose and tested three Master Mixes which meet our demands as following.

a. Fast Cycling PCR (Qiagen)

b. Phusion High-Fidelity PCR Master Mix (Thermo Scientific)

c. Phusion Flash High-Fidelity PCR Master Mix (Thermo Scientific)

Evaluation

a. Fast Cycling PCR (Qiagen) : Basically it was good, but poor yields for 1kb or longer amplicons found, even followed the instruction. There is not the 2-step protocol available in the instruction. One hour with 35 cycles. The most asset in this master mix is that almost of reactions work on the same melting temperature, at 60 degree C.

b. Phusion High-Fidelity PCR Master Mix (Thermo Scientific) : The 2-step protocol did not work for us. The 3-step protocol takes over 1 hour, so that it does not meet our demand.

c. Phusion Flash High-Fidelity PCR Master Mix (Thermo Scientific) : Basically it was as good as the Qiagen’s, and better yields for longer amplicons found than Qiagen’s. On the other hand, the 2-step protocol did not work for us as well. One hour with 35 cycles. We need to optimize the melting temperature on demands and this point is not as good as the Qiagen’s.