ganesan at milton.u.washington.edu
Fri Mar 20 13:24:31 EST 1992
With respect to gene disruption in coli, I remember that at one point
people were using pBR322 derivatives with a similar construct to yours but a
bacteriostatic antibiotic resistance gene in the middle ( CM would do nicely
but tet might be easier for subsequent stps as described below )
and put the plamid into a polAts strain. ColE1 replicons need polI and at 42
the plasmid integrates homologously ( in its entirety ). Once you have got
the TetR colonies isolated you then select tets derivatves with the standard
Bochner or Malloy methods for tet sensitivity. The plasmid will excise one of
two ways leaving either the intact gene or your disrupted gene. The frequency
should be about 50/50. I got about 40% of the kind I wanted when I did it
a few years ago.
The other option is to transform in linear plasmids and hope for RecBCD
to do its job. But that frequency would be lowered significantly if your
flanking regions do not contain Chi. RecBCD would also degrade your incoming
DNA quite readily. In a RecBC - background Graham Walker described a protocol
for doing just this. For references on polI stuff look for Cox or Gibson's
papers around 89 or 90. If you cant find them write to me and I will scout
them out for you. For Chi RecBCD stuff look for Gerald Smith's papers( he
happens to be the one I work for and this account was set up on his budget)
and I am pretty certain you can find Walker's paper quite easily.
sganesan at fred.fhcrc.org
ganesan at u.washington.edu
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