poor cloning efficiency w/Pfu turbo

Roland Hubner roland.hubner at ua.ac.be
Wed Jul 31 08:39:40 EST 2002


>> After PCR, they are sodium acetate precipitated and then digested
>> with Sfi overnight.
> 
> Taq survives Ethanol pptation and some nucleotides also ppt.
> 
> Pfu may survive even better.
> 
> You then land up with a nice gap filling enzyme during your overnight 
> Sfi I digest so your overhangs get blunted hence lower cloning 
> efficiency.
> 
> The survivability is documented by Wayne Barnes in Gene several years 
> ago in his Blue/white fidelity assay for KlenTaq.

 Alternatively, supposing equally clean rxn's and no error in the Sfi 
recognition sites, could the "turbo"-factor be blamed for wrecking the 
SfiI-ends?

 Finally, Sfi I requires _TWO_ recognition sites in /cis/ for optimal 
activity... ref. given in NEB catalog, p. 246 (2002-3)

 R.



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