RT-PCR problem

Jeremy Coate via methods%40net.bio.net (by jec73 At cornell.edu)
Fri Feb 2 11:27:29 EST 2007


A couple quick thoughts:
1.You might have an inhibitor of the PCR reaction in your cDNA that is
sufficient to stop the taq at 4ul but not at 2ul. I'd try using less
template and see if that helps.
2.Have you confirmed that your PFU taq works with other templates?
3.Are you giving the enzyme the necessary hot start heat cycle to activate
it at the start of the PCR run?

I haven't worked much with PFU, so there are probably other possibilities
specific to that enzyme that I'm not aware of.

Jeremy


On 2/2/07, 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
>
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