PCR mistakes

Koen De Smet k.desmet at nospam.ic.ac.uk
Thu Jun 4 01:54:44 EST 1998

Richard P. Grant wrote:
> In the first instance, simply Taq, yes.
> I've used the same idea for Pfu too.  Taq I've tended to do 25 cycles, Pfu
> 30 cycles.  Taq was a home-made buffer, Pfu (cloned) was the supplied, but
> I've always titrated Mg2+ concentration.
> It's a bit worrying that you're getting errors using a proof-reading
> enzyme.  I would suggest trying different enzymes.

I have used Deep Vent (NEB) to amplify and clone several ORFs from Mycobacterium 
tuberculosis, and found no seqeuncing errors up to now.
> Silly question - how do you know you have the mistakes?  Are you comparing
> with a published sequence?
Maybe not so silly. In one of my clones, I had some differences from the sequence in 
the EMBL/GenBank database. But when the genome of M. tb strain H37Rv was sequenced 
(finished last December BTW), this ORF was identical to the sequence I had from my 
amplified product, but different from the original one in the database.

Koen De Smet
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     Imperial College School of Medicine at St Mary's   

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