dTTP -> dUTP problems in PCR

Richard THIERY rthiery at ifremer.fr
Wed Mar 4 09:20:55 EST 1998



Nico van Belzen <belzen at hema.fgg.eur.nl> a écrit dans l'article
<6dja4b$r8$4 at popeye.eur.nl>...
> Hi all, 
> 
> (posting this for a colleague) 
> In order to prevent contamination, I want to use dUTP/UDG (uracyl 
> glycosylase) in all diagnostic PCR systems. However, sometimes the 
> reaction is not working when I change from dTTP to dUTP. I tried to 
> increase the dUTP concentration, this did not help. Trying other 
> brands of Taq polymerase does not work. Does somebody know a solution 
> for this problem? 
> 
> Best regards, 
> 
If you are using UDG before doing the PCR reaction make sure that you
inactivate properly this enzyme (10 min @ 95°C), otherwise any residual
activity will destroy your PCR products. Also it is better if the annealing
temperature of your primer pair is above 60°C. Try also adding an extra 5
cycles to your PCR because you may have a decrease in PCR efficiency any
way (eg 40 cycles instead of 35 cycles).
Hope this helps, good luck.

Richard THIERY            
CNEVA/LPAA 
Technopole Brest-Iroise, BP 70
F-29280 Plouzané  FRANCE



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