PCR problem

[Heavy E]

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 
follows:
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 
can.	
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...


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