Bp-exchange by PCR

vilimf01 at mcrcr6.med.nyu.edu vilimf01 at mcrcr6.med.nyu.edu
Wed Dec 11 12:43:22 EST 1996


This has happened to me as well.  Pfu had a 5-12 fold lower error rate
as compared to taq, and stratagene says you can expect 3-4% of your
clones to contain errors.  The other places that your errors could
come from are - 1) If you are gel purifying, the UV can introduce
mutations, and 2) If you are using a plasmid as a template, the insert
could have mutated somewhere along the line (a sure fire sign of this
is if more than one Pfu clone contain the same mutations).  Good luck.

					Regards,	SVEN

In article <32AD2B0D.3E5D at urz.uni-heidelberg.de>, Klaus Lun <klaus.lun at urz.uni-heidelberg.de> writes:
> I have amplified a 1.2 kb fragment with Pfu Polymerase and after
> sequencing I found 2 Bp-exchanges, leading to  aminoacid substitutions.
> So I was wondering if this is the 'normal' mutation rate you get with
> Pfu-Polymerase; because I want  to express this fragment I absolutely
> need no Bp-exchange. So how many PCR-poducts should I clone ? 10, 20 ? I
> perform the PCR with 30 cycles ..
> So please, help me ..........
> 
> 	KLAUS



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