PCR problem

Dr. Duncan Clark Duncan at nospam.demon.co.uk
Tue Feb 6 12:51:57 EST 2001


In article <F14HL2bti2SbPkPuOlv00014f46 at hotmail.com>, the eminent divya
venugopal at BIOSCI/MRC Human Genome Mapping Project Resource Centre
wrote
>I'm trying to amplify a 1.5kb region from the origin of replication of a plasmid 
>from Mycobacterium avium-intracellulare.  The primers have been designed from 
>the published sequence of the origin of replication.  The calculated annealing 
>temperature of the primers is 67 degree C.  However i did not get any results at 
>that temperature.  I got an amplicon at 55 degree C.  The size works out to be 
>0.5kb.  What could this be as i should be getting a 1.5 kb band. How is it 
>possible to get the expected band.

Are there a lot of repeats around the origin such that the primers are
having difficulty annealing to the right place?

If there are repeats could the enzyme be mis-reading?

Is the PCR product severely AT rich. If so you could be actually melting
it at your 67C or 72C extension temp. If so you will need a lower
extension temp. 

Is it GC rich? If so try betaine at 1M final in your PCR.

Have you tried a Taq:proofreading pol mix in case Taq or Pfu or whatever
you are using doesn't like amplifying the PCR product due to one of
above?

Have you tried Mg optimisation in 0.5mM steps from 1.5mM up etc?

Just a few things to think on.

Duncan
-- 
The problem with being on the cutting edge is that you occasionally get 
sliced from time to time....

Duncan Clark
GeneSys Ltd.
Tel: +44(0)1252376288
FAX: +44(0)8701640382
http://www.dnamp.com
http://www.genesys.demon.co.uk






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