PCR mistakes

[Zophonias O. Jonsson]

In article <6l3hkj$clk at sun0.urz.uni-heidelberg.de>,
un691cs at genius.embnet.dkfz-heidelberg.de (Clemens Suter-Crazzolara, PhD)
wrote:

> On Wed, 03 Jun 1998 12:20:45 +0100, Richard P. Grant said:
> 
> >It's a bit worrying that you're getting errors using a proof-reading
> >enzyme.  I would suggest trying different enzymes.  
> 
> Yes, that was one of my ideas too. Perhaps I should try Pfu like you did.
> 
> >Silly question - how do you know you have the mistakes?  Are you comparing
> >with a published sequence?
> 
> You address another good point. The sequence was published long ago, I
have amplified 
> it without apparent mistakes in the past, but now all of a sudden I find
> PCR errors. So, I thought it were maybe sequencing errors, but the ABI
profiles
> look pretty convincing, and the mistakes appear to be distibuted
> more or less throughout. I still haven't figured this out completely.
> 
> 
> clemens

What kind of mistakes are you getting?  Could it be that your dNTP mix has
gone bad?  Oxidized dGTP can mispair quite effectively and give rise to
mutations.  The oxidation would have to be quite severe to account for
four mistakes in 500 bp so it's probably not the only reason, but it may
be worth trying a fresh batch.

Zophonias

_____________________________________________________________________
Zophonias O. Jonsson
Institut fur Veterinarbiochemie               Tel: (41-1)-635-54-75
Universitat Zurich-Irchel                     Fax: (41-1)-635-68-16
Winterthurerstrasse 190
CH-8057 Zurich
Switzerland
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