RT-PCR problem

Jose de las Heras via methods%40net.bio.net (by josenet At tiscali.co.uk)
Fri Feb 2 14:19:57 EST 2007


"Cuifeng Yin" <cuifeng At gmail.com> wrote in message 
news:mailman.1039.1170432668.19683.methods At net.bio.net...
>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

If you can't easily troubleshoot your Pfu PCR, why don't you go ahead with 
Taq anyway?
In my experience you do get some mutations, but not that often. I have 
cloned stuff of up tp around 1kb from common Taq PCR. Just sequence a few (a 
handful only) of the clones, and I'm positive you'll find at least one that 
looks okay. Then re-sequence to be absolutely sure. It's a 3-day job max 
(assuming you do get your sequencing done in-house, like I do).

Jose 




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