RT-PCR problem

Cuifeng Yin via methods%40net.bio.net (by cuifeng At gmail.com)
Fri Feb 2 04:40:30 EST 2007


I am having RT-PCR problem. After RT, I did PCR using Qiagen Taq and
Promega PFU ultra HotStart. For qiagen Taq, it gives me a nice band at
~1.3 kb which is the correct size. However, for PFU, I got nothing. I
set up both PCR reactions according to PFU suggestions. The only
difference between the two reactions are enzyme and buffer. And for
PFU PCR, I used 4 ul of RT reactions, which is twice of the amount I
used for Qiagen Taq.

Anybody has any idea why this happened? Since I need to express the
gene in E. coli, I can not use the product from Taq, which tend to
give many mutations.

Any advice will be greatly appreciated.

Diana



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