Rules of thumb for sequencing cloned PCR products?

Bryan John Maloney bjm10 at newsstand.cit.cornell.edu
Thu Aug 29 19:48:32 EST 1996


Mark Pallen (m.pallen at ic.ac.uk) wrote:
: I remember reading somewhere that a rule of thumb was that you are 
: likley to get one misincorporation error for every 1.5 kb that you 

It may be a rule of thumb, but I know that an amplification that a post-doc
in our lab did was worse than that.  She used a mixture of Taq and Pfu and
still got two deletions.  We know that they're deletions because she was 
amplifying a synthetic sequence originally assembled from oligos.  
Furthermore, a different clone from the same amplification ended up not
having the deletions.  The deletions were confirmed by one machine and
two "by hand" sequencings.

: doing three separate PCRs and cloning then sequencing all three was a 
: reasonable way to get round the Taq/PCR-induced error problem. My 

I would say that, if all three agree, then you should be okay.  However,
I wouldn't expect all three to agree.

Sorry I can't be less anecdotal, but this is a fresh event.




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