poor cloning efficiency w/Pfu turbo

Paul Shinn pshinn at mail1.sas.upenn.edu
Wed Jul 31 12:30:35 EST 2002


: As others mentioned, carryover of Pfu and some nucleotides can be an 
: explanation. Another, more likely, IMHO, is that Sfi works/survives
: better in Taq buffer than in PfuTurbo. 

: In any case, why not use silica-based PCR cleanup (like Qiagen?).
: With manifold and money to buy kitsd you can do 24 samples in 
: about 5 min. Beats EtOH pption any time and kills/gets rid of Pfu 
: 100% sure. For ligation, heat killing is usually sufficient, no need to 
: pptate or purify. 

   We precipitate after PCR and then digest in Sfi. Wouldn't the Taq/Pfu 
buffer components be washed away after precipitation?
   We have Millipore filter plates we were going to use for cleanup of 
our sequencing reactions.  They use Sephadex G-50 resin.  This won't 
get rid of/inactivate the Taq/Pfu will it since the cutoff size is 5000 
Daltons?

				Paul



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