Ligation, and Transformation iNHIBITORS.

Bernard P. Murray, PhD bpmurray*STUFFER* at socrates.ucsf.edu
Mon Mar 8 23:11:17 EST 1999


In article <B0000112210 at spider.cigb.edu.cu>, amherrera at CIGB.EDU.CU wrote:

>     Hi all
>     I having a low transformation frequency when transform my 
>     ligation? 
>     I have evidence about the good efficiency of the competent 
>     cells.
>     The vector I used is 10.kb and the band over 3.3kb
>     Thanking Any Suggestion   
>     D.Q.V

I doubt if its a problem with inhibitors.  Ligate overnight
at 16degC and then transform as normal.  The efficiency is
really pitiful using chemical transformation (Inoue et al.
cells, heat shock) with big plasmids.  I've had to try this
with some old yeast vectors (eg. >12 kb) and knew there was
no problem with the ligation mixture or the competence of the
cells.  You can try playing with different heat shock times
but at the end of the day its best to do several ligations
and transformations and spread them over lots of plates.
If you can't boost the proportion of your recombinants
(eg. cutting the ligation) then bite the bullet and go for
colony hybridisation.  Alternatively, use a high throughput
screening method such as direct colony PCR.
     I don't know if electroporation is a better choice for
big plasmids.  Perhaps someone else can comment.
     Use a smaller vector if you can....
          Bernard
-- 
Bernard P. Murray, PhD
Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA



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