A.Rosin wrote:
> Hi,
> I wonder if any of the Li-Cor users out there can give me some help.
> I keep getting what looks like unspecific termination on my gels. It
> looks
> particularly bad on the pGEM samples. I get 60-80 bases of nice
> material,
> then a blob of dye across all four lanes, then a really weak signal which
> finally dies out. I add DMSO to my gel mix and the PCR reaction, I've
> tried
> increasing cycle number and elongation times, but it has had little
> effect.
Do you get a big fat wavy blob of dye (also known as the Northern Lights
effect) or is it just one band (termination) across your lanes? If it's
termination, I've found that the quality of the template DNA plays a large
role. I sequence PCR products and I used to gel purify them, but even a
little exposure to UV light would nick it enough so it wouldn't sequence
very well at all.
You might also want to call or e-mail the folks at Li-Cor, I've found them
to be very friendly and helpful.
Good luck-
Gina
