Help!PCR problems.

tina guina tguina at wesleyan.edu
Fri Feb 24 10:51:01 EST 1995


In article <1995Feb15.135846.41809 at cc.usu.edu>, sl1nn at cc.usu.edu wrote:

> I am having problems with PCR amplification of a small fragment of
cloned DNA.  I
> I have designed my own primers, using Oligo 4.1, and according to the
program I
> am using the right temperatures for the primers. I have varied tha conditions
> several times with the same results.  The negative control (without
> template) of my two primers always shows a 250 bp band.  Both primers are
> 22-mers and the dimers would not be that large. In additon, when I have the
> template present I am only seeing the same band. I have checked the
primers for
> contamination using HPLC, and they check out at the right size and with only
> one peak.  Does anybody have any idea why I am getting this result?  I'm going
> crazy trying to fix the problem.   Thanks in advance.

Problem is actually very simple- there is no problem at all- you don't
need to waste your time doing HPLC or any of other fancy stuff- just use
noncontaminated, fresh solutions, and it is very useful, we found to use
pipet tips with filters (as for work with radioactive isotops). It seems
that even if you use sterile tips w/out filters that pipetman transfers
DNA in traces (as aerosol) to another, negative control tube- and there
comes your band from!
We had similar problems in the lab and, just by changing tips, we eliminated it.
Tips are called "ART", and can be ordered from FISHER.

Good luck, T.



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