help PCR

J Kennie 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
> ---
> julian.robinson at plant-sciences.oxford.ac.uk
> 
> 
Julian,

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. 
Help.

Jan 
University of Alberta




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