RT-PCR problem

Brian Ballard via methods%40net.bio.net (by NoReply At email.com)
Fri Feb 2 15:51:25 EST 2007


In article <mailman.1039.1170432668.19683.methods At net.bio.net>,
 "Cuifeng Yin" <cuifeng At gmail.com> wrote:

> 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

Try adding 10% DMSO to the Pfu reaction or altering the annealing 
temperature. You'll find that some templates amplify great with one 
enzyme but not with another and there's not usually a simple explanation 
why. Adding DMSO and changing annealing temperature is usually where I 
start.

Brian


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