Howard R. Smith (HRS at levin.cri.nz) wrote:
: Hi netters,
: I'm looking for a method for getting good reliable plasmid preps
: from Agrobacterium. In our lab we can get good plasmid preps from LBA4404,
: A722, A4, and 9 other strains if they contain a pGA643-based plasmid or
: pKIWI110. We can't get any reliable result if we try A281, EHA101, or C58,
: or if we try LBA4404 or A722 containing a pBIN19 or pBI.121-based plasmid.
: Any suggestions?
: Speaking of pBIN19-based plasmids, how do you transform them into
: Agro and then get the sodding things to grow? We find that a zapping in BRL'
: s electroporator followed by 1-2 hours in YT or LB media before plating onto
: selective media works fine for other plasmids but even mention the word "
: BIN19" and things die.
: Finally, in our hands we find that LBA4404 has a propensity to
: insert a fragment of >2kb into pGA643-based plasmids from time to time. Has
: anyone else ever encountered this problem?
: Either E-mail me direct or better still post back here and start
: some discussion going!
: Howard Smith
: Plant Pigments Group
: New Zealand Institute for Crop & Food Research
: New Zealand
:SMITHH at crop.cri.nz
I have used a modified alkaline lysis procedure with Agrobacterium
(C58,LBA4404,LBA4011,LBA4301) which starts with a ten minute lysozyme
treatment at 37 oC. The rest of the procedure is essentially according to
Maniatis except I use 300, 600, and 450 ul of the first three solutions.
Lysis is still not as good as with E.coli and some chromosomal
contamination is present, but you can see even low copy plasmids.
The problem with pBIN19 is likey that it is being restricted upon
Mike Cooley mbcooley at ucdavis.ucdavis.edu