Primer Extension

Cornelius Krasel krasel at alf.biochem.mpg.de
Sat Nov 26 05:26:14 EST 1994


Claudia Salinas (salinas at bcu.ubc.ca) wrote:
> I am looking for protocols for primer extension other than
> that in Maniatis.  I am a primer extension novice, so any tTrouble
> shooting
>  suggestions or advice would be appreciated.

What's your problem? I found the "Red book" protocol quite reliable
(Current Protocols in Molecular Biology), with one exception:

The oligo is hybridized with the RNA overnight. To get quantitative
results, it is extremely important (in my experience) to prewarm the
tubes to 42oC before adding the other compounds (Tris, MgCl2, DTT,
dNTPs, RNasin and AMV). I spent about half a year trying to get
reproducible bands before I found this out :-(

--Cornelius.

--
/* Cornelius Krasel, Abt. Lohse, Genzentrum, D-82152 Martinsried, Germany  */
/* email: krasel at alf.biochem.mpg.de                 fax: +49 89 8578 3795  */
/* "Science is the game you play with God to find out what His rules are." */



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