jkennie at GPU.SRV.UALBERTA.CA
Fri Oct 3 13:12:39 EST 1997
On 1 Oct 1997, Julian Robinson wrote:
> Iam trying to amplify a ~450bp fragment with two 20bp primers.
> It was working perfectly before I went on holiday. However when I
> came back I begtan to have problems. The problem was smears in all
> the lanes, including the control lane. It wasn't the template DNA
> that was the problem (it oocured in the -ve control), and it is'nt
> contamination (new everything has been used).
> We are leaning towards primer dimers as the answer at the moment (as
> there is some complementarity? between the primers), but the real
> question is why has it just started to occur, when it was perfect
> (i.e. no smears and clean bright product) before I went on holiday?
> many thanks
> julian.robinson at plant-sciences.oxford.ac.uk
I have been having the same problem. My template should be fine, the
primers I use are kept at -20, I've tried new buffers, enzyme, water,
dNTPs, etc. and I still get smears in all the lanes. Incidentally, this
started happening a few days before I went on holiday and is still a
problem now that I'm back.
Does anyone have any suggestions on how to fix this problem??
I am amplifying a 850 bp fragment using primers that are about 30 nt long.
I have previously had it working fine.
University of Alberta
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