ratnakar amaravadi amaravad at copper.ucs.indiana.edu
Sat Apr 18 23:00:09 EST 1992

Dear netters, 

We are facing a serious problem in our lab with template contamination
in PCR reactions.  Anybody's ideas regarding where the contamination
is coming from will be greatly appreciated.

Situation follows:

When we do a no template control with a set of oligos it gives the
right band and even the intensity on an agarose gel is same as a
reaction with the template.  We checked all the possibilites for
contamination including Taq buffer, Mgcl2, dNTPs, water and the
mineral oil.  Reactions have been repeated many times and we figured
that it was not from any of these reagents.  Thinking that we have
traced the contamination to the oligos, and with no other way to go we
have made new sets of oligos (spanning different regions of the
gene) and again all the controls showed that our new oligos are
contaminated again (during purifications in the lab?). This time we
thought it was coming from the pipettes we use because we use the same
pipettes for other purposes in the lab.  We made a  third set of
oligos again, and these were purified carefully with all new buffers
and this time we used plugged pippet tips.  Again after eliminating
all the other possible sources of contamination in the reaction,
oligos seem to be contaminated.
	Is it possible that the contamination is coming from the air?
If anybody can think of something or if you have any ideas about how
to get around this, please reply by e-mail to amaravad at copper.ucs.indiana.edu.
or in the net. 

Any comments will be greatly appreciated.

Thanks in advance.

Lakshmi Amaravadi
Dept. of Life Sciences
Indiana State University

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