nested PCR

Elizabeth Ojeda eojeda at gema.org.ar
Thu Mar 19 08:27:20 EST 1998


In my experience, the step most dangerous for contamination is to pass from
round 1 to round 2, so you must use opener tube for each tube. I think this
action is strong preventive. I use the opener tube from Robins Scientific. I
dont´t know if it exist other company that to buy it.
Hope this comment help you.
Elizabeth
eojeda at gema.org.ar
-----Original Message-----
From: Ulrich Maier <maier_hml at muenster.netsurf.de>
To: methods at net.bio.net <methods at net.bio.net>
Date: Miércoles 18 de Marzo de 1998 09:34
Subject: Re: nested PCR


>"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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