nested PCR

Ulrich Maier maier_hml at muenster.netsurf.de
Wed Mar 18 06:44:12 EST 1998


"Richard THIERY" <rthiery at ifremer.fr> wrote:

>Hi all,
>
>Any suggestions to prevent false positive results in nestedPCR reactions? I
>am trying to set up such a test to detect a fish virus. Sometimes I get
>some positive  reactions in my blank controls. Its seems to be due to carry
>over products from the first step because I have no problem with the first
>step controls.
>Many thanks for your help
>
>Richard
>rthiery at ifremer.fr 


Hello,

try a one-tube nested PCR with trehalose-lyophilized Taq. The method
is described in:

PCR Methods and Applications, Cold Spring Harbour Laboratory Press,
pages 376-379.

Works fine. Advantages: Prevents crossover contaminations and  you can
prepare a lot of tubes with the same PCR efficiency. You can store
prepared tubes at 4°C.

Disadvantages: You have to work with a mineral oil layer without a
heated lid, and if you have problems preparing the tubes it is getting
expensive.

Hope this helps, 

Uli Maier 



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