RT-PCR problem

Pow Joshi via methods%40net.bio.net (by pow.joshi At gmail.com)
Fri Feb 2 15:19:54 EST 2007


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.

am wondering if you Mg+2 concentrations were higher than 2mM ....it
seems to me that the 10X reaction buffer already has 20mM MgCl2,
however, if you would wish to try the reaction adding another 1mM of
MgCl2......
 I suppose for cDNA amplification atleast 3mM Mg+2 is required ( or so
the stratagene manual says).....

best
pow


>
> Any advice will be greatly appreciated.
>
> Diana
>
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