cloning pcr from VENT,Pfu etc.

John Nash nash at nrcbsa.bio.nrc.ca
Mon Nov 8 12:14:59 EST 1993


In article <9311080531.AA12832 at qimr.edu.au>,  <rogerM at qimr.edu.au> wrote:
> Dear clonetters, 
>     Does anyone have experience cloning pcr fragments from the other
>   thermostable polymerases particularly VENT and DEEPVENT from NEB 
>   and PfuI from Stratagene. What successes and/or failures have people

Since VENT leaves blunt ends, it's been my enzyme of choice, for
amplifying fragments to clone.  Amplify, cut out of gel, clean with
glass, and clone into blunt-cut vector of choice.  

I think DeepVent (and Pfu) leave single end overhangs.  Since my
experience with the TA kit was entirely a waste of time and money, I'd
recommend incubating amplified fragments with T4 DNA polymerase for 5
min (add 1 ul enzyme to amplified DNA, incubate on bench for 5 min,
DON'T heat kill - it seems to wreck the DNA, cut out of gel, clean
with glass and clone into blunt-cut vector).  I haven't tried this
with Pfu, I can't recall whether I tried this with Deep Vent, and it
works nicely with Taq.

>   experiences. Also has anyone had problems cloning pcr fragments
>   using degenerate primers containing Inosines. We have had one

No. but I've got it to work with degenerate primers.  I screened
several clones, each seemed to have the correct insert (from
sequencing internal sequences), but the primers were not always
correct matches, indicating amplification of the correct fragment from
mismatched primers.

>   success with these primers but other different  degenerate primers
>   have been totally unsuccessful. There are white colonies showing
>   up but the majority show up to be deletions if >100bp in the insert
>   and unsequencable due to apparent deletions in the polylinker. The
>   fragments were tested against control fragments from specific pcr
>   primers by self-ligation and the test set totally failed whilst the 
>   controls all self ligated.Any tips, suggestions or spelling flames

With 3 or 4 of my cloning attempts with amplified DNA, the correct
recombinant was a blue colony!  Screening clones with labelled
amplified fragment may be a nice option.


-- 
John Nash                           (nash at nrcbsa.bio.nrc.ca)
Institute for Biological Sciences,  National Research Council of Canada,

      *** Disclaimer:  All opinions are mine, not NRC's! ***



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