poor cloning efficiency w/Pfu turbo
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
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