Deletion of plasmid

Bernard Murray bpmurray*STUFFER* at socrates.ucsf.edu
Wed Jul 15 18:35:20 EST 1998


In article <rXxS0AAEXHr1Ewho at demon.co.uk>, "Dr. Duncan Clark"
<duncan at genesys.demon.co.uk> wrote:

> In article <35AC33BA.2B0A at bioneer.kaist.ac.kr>, Jung Hoon Ahn
> <jhahn at bioneer.kaist.ac.kr> writes
> >But strangely, after ligating two, transfoming it into E.coli XL1-Blue,
> >mini-prepared plasmid is not what I expected. The front part of gene is
> >deleted.
> >I inserted the rest part of gene into the cloned gene in pUC19.
> >What is the reason of this phenomenon and how can I solve this probelm.

> If the gene is expressing and lethal to E.coli then the E.coli will
> either die or the gene will be deleted. Try cloning opposite to the lac
> promoter, use pBR322 or a lower copy vector, don't clone it with a
> promoter etc.
> Duncan

I can echo Duncan's advice as I also had problems with an ABC
protein.  Make sure that the F' (and thus the Iq) is present
in the XL1-Blue (I think this requires tet selection)
and do not use blue/white selection with the construct
(you can screen by PCR or restriction digestion of minipreps).
Both these conditions should reduce leaky expression from
the Lac promoter.  For a handy non-Lac plasmid try one of Promega's
old pSP plasmids (eg. pSP64).  If you think the bugs are part of the
problem then maybe try HB101, SURE, TOPP etc.  You could also
try changing the culture conditions (richer broth, lower temperature
etc.).
     Good luck,
          Bernard
-- 
Bernard Murray, PhD
Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA



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