PCR Woes.

Sergio sergioal at bbm1.ucm.es
Wed Aug 15 05:33:10 EST 2001



"Mr. John P Phelan" wrote:

> Dear All,
>
> Apologies for my lack of info in my previous e mail. Here's the specifics.
>
> I am trying to PCR up a 3.5 kb fragment from a plasmid using pfu.
>
> Basically I am seeing alot of smearing at the
> top of my gel, most probably template.

You shouldn't see the template (or at least not "a lot") in the gels since a
very low amount of it it's more than enough. In my opinion, strong smear is
ssDNA produced by the elongation from one (or both) of the primers, and either
the distance between the primers is longer than the polymerase capabilities, or
one of the primers is not annealing properly. To check whether both primers are
annealing where they are expected to do, you could sequence from both of them or
just perform two control PCRs using only one primer, and checking for the
absence/presence of ssDNA. However, this last method only proves that the
primers are annealing, but you won't know where they are doing it.
I had a lot of problems using Pfu on long product PCRs. Finally i had to use Taq
and substitute the mutations by replacing some internal fragments by
restriction.

Sergio






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