PCR Problems -- Help?

Rafael Maldonado rafael at howard.genetics.utah.edu
Fri Sep 29 18:53:59 EST 1995


On 29 Sep 1995, James Chen wrote:
> 
> Have any of you done controls for your PCR reaction with same strand primers
> and produced amplification?
> 
> I'm pretty sure that there isn't any contamination with template (ie, new
> batch of oligos produced the same amplification band.

It's very difficult get rid off contaminat DNA. It is everywhere, and 
PCR is very powerful detecting it.

I suppose you have used totaly different stocks of everything (autoclaved 
if possible), used different set of pipetes, and so (those plugged tip 
are very useful). In that case, you are in the same situation than I was 
several years ago:

Some oligos gave me a band even I knew for sure that there was no target 
in my template. The band was the right size, so I spent several days 
cleaning everything, even going to other floor to repeat the experiments.
The band persisted. 

Finally, very well advised, I made a Southern to see if that band was the 
same that in the positive control. Surprise! No signal in the hybridisation!

The band was exactly the same size (a unspecific amplification), but was 
not homologous at all with the sequence I was trying to amplify. So try a 
Southern. If your band is a unspecific amplification, or a real 
contamination, you will no what to do...


	-Rafa

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                                             |
Rafael Maldonado                             |  La cita ha sido
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