PCR problem

Heavy E ez022056 at peseta.ucdavis.edu
Wed Sep 27 12:52:26 EST 1995

here's one for the PCR experts...
I am trying to do a pcr reaction which requires a 72 deg. soak (3') 
before the addition of TAQ.  What I would do is let the machine run the 
3', 72 deg. pause it, add the taq, and unpause it.  The parameter is as 
72 deg, 3'
72 deg, 5'
95 deg, 1'
72 deg, 3'
back to 95 deg for 20 cycles
72 deg, 10'
4 deg soak.
The problem iswhen I do one tube of this 
reaction, the product looks fine.  But, when I do multiple tubes (5 and 
7), I get nothing.  I've tried to make the technique as consistent as I 
Keep in mind the parameter doesn't appear to be the problem because I did 
get the reaction to work, using the same parameter.  And doing all the 
tubes individually is not a likely option because I need about 30 tubes 
of this stuff.  Also, I am doing 200ul reaction in the small variety of 
PCR tube...is that going to be a problem?
Thank you...

Edward H. Wang                 *
Staff Resch. Assc.             *
Dept. Internal Med. -Nephrology*
UC Davis                       *

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