Problem PCR

Dr. Duncan Clark Duncan at nospam.demon.co.uk
Thu Apr 6 05:36:59 EST 2000


In article <38EC56D0.F738977C at galton.ucl.ac.uk>, nntp-server-
test.ucl.ac.uk <joanna at galton.ucl.ac.uk> writes
>I am trying to PCR across a tandem repeat region which is highly GC rich
>(approx 82%).  The repeat region is asymmetrical in that one strand
>contains mostly Cs and the opposite strand mostly Gs.  The repeat unit
>varies in size from 2.5-5Kb with most of the population having one small
>and one large allele.  All I get when I try the PCR is the smaller
>allele and lots of smear.  I've tried increasing the annealing temp,
>extension temp, increasing the extension time, increasing the
>denaturation time and temp.  I've tried adding glycerol, DMSO, and
>betaine but all to no avail.  Does anybody have any tips or tricks I can
>try?
>

Try using a polymerase mix i.e. Taq plus a proof-reading pol. Add in
DMSO to 5-10% and Betaine to 1M final. You may also need to raise your
denaturation temp by 1 or 2C, maybe up to even 98C. Unfortunately that
will have a nasty effect on the stability of the Taq component so make
sure you run minimal denaturation times i.e. 5-10 secs max.

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

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




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