PCR problems

Dr. Duncan Clark news at hgmp.mrc.ac.uk
Thu Aug 16 07:52:42 EST 2001


In article <24edbbf8.0108160039.6b52cce7 at posting.google.com>, the
eminent vibeke at http://groups.google.com/ wrote
>I am using universal primers and the
>PCR conditions are: annealing 56*C, 1 min. and elongation 72*C, 3.5
>min.

I'm not surprised given universal primers. I would reduce the annealing
time to 15 secs and the extension time to 1-2mins, possibly reduce the
primer concn. and then run a cycle no optimisation i.e. run an aliquot
out on a gel after 15,17,19 .... etc cycles and see when the smear is
visible.

Possibly playing with the Mg concn and annealing temp would improve
things.

On the other hand if you are PCRing a complete genome set of already
generated PCR products then you will only see a smear. Whether that
smear is correct depends upon the size range you see.

I take it that the universal primers don't match anywhere against the
genome itself.

Duncan 
-- 
Homogeneous Fluorescent Reporting Systems for Real-Time Quantitative PCR: 

Optimisation, Probe Technology & Future Systems

4-5 September 2001
King Alfred's College, Winchester, UK 

http://www.dstl.gov.uk/html/specialisations/index.htm




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