PCR failing

LeboMad via methods%40net.bio.net (by lmadubanya from gmail.com)
Thu Apr 8 14:23:54 EST 2010


On Apr 7, 2:58 pm, d... from no.email.thankstospam.net (DK) wrote:
> In article <5a4d0356-fa5d-45ea-8b98-4b1eed90b... from u31g2000yqb.googlegroups.com>, LeboMad <lmaduba... from gmail.com> wrote:
>
> >Hi all. I have a serious problem with PCR amplification in my lab. I
> >have done all troubleshooting there is out there I can't seem to get
> >it right. I normally get nice clear bands but for some reason I get
> >very faint bands if any at all. Can anyone suggest anything. I've got
> >a student that needs some PCR runs and needs result as in yesterday.
>
> Imagine a student coming and telling you that "nothing works, please
> help me" - without any pertinent details. Would you be able to help?
>
> DK

ok everyone thanks for responding and sorry for responding late. I am
working on a multiplex PCR where the annealing temperature of the 3
primer sets has been optimized to work at 58. The products I get are
920, 800 and 700 bp.  Optimum DNA conc is 50 ng, 2mM MgCl2, 2mM dNTP.
I use 30ng primer concentration. Profile thus is 94 C 5 min (1 cycle),
94 C 1 min, 58 C 1 min, 72 C 1.5 min (x 35 cycles) and 72 C 5 min. I
separate fragments on 2% agarose. I use Promega Taq.

My frustration is that 2 months ago this was working perfectly. Lately
if I'm lucky I get very faint bands or none at all. What came to my
attention was the primer dimer formation very evident on the gel. My
student actually thought for some time that the dimer was the result
without even confirming the size with the ladder.

any ideas???

Thanks
Lebo from South Africa


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