Jose de las Heras
(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.
If you can't easily troubleshoot your Pfu PCR, why don't you go ahead with
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).
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