poor cloning efficiency w/Pfu turbo

Paul Shinn pshinn at mail2.sas.upenn.edu
Fri Aug 2 13:15:06 EST 2002


: Sfi I is incubated at 50C and even though Pfu is a lot less active and 
: slower, an overnight incubation at 50 you only need 1bp adding or 
: cleaving away to lower the transformation efficiency. As the original 
: contributor was seeing a 5 fold lowering that isn't actually a lot in 
: the whole scheme of things. Maybe 0.001% of enzyme surviving would be 
: sufficient to do this :-((

: I think the outcome is to treat the PCR products properly and then if 
: the cloning efficiency is still down for Pfu we need to think of another 
: excuse.

    Thanks for the all the responses.  Maybe it's just a bad week for 
cloning.  Anyway, this is what I've resorted to:

   Since Sfi is leaving a 3 base overhang, I'm thinking Pfu has either 1) 
filled it in or 2) chewed off the overhang.  I incubated my PCR product 
with Taq and dATP, then TOPO-TA cloned it last night.  I got clones!  I'm 
going to sequence my clones and examine the junction between the TOPO 
vector and the PCR product.  I'll see if the overhang has been filled in 
or chewed back.

						Paul



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