ihanvan at imvs.sa.gov.au
Tue Jun 22 19:13:33 EST 1999
Is your donor lambda pir +,
I have done a lot of conjugation in my formative years with S17-1 L-pir
moving pir protein dependant plasmids bearing Mini-TN5 reporter constructs
I know of 2 basic methods for conjugation (provided your using the right
1.. Liquid Culture
2.. Grow overnight cultures of both donor and recipient in suitable
3.. Mix donor and recipient in 1:10 ratio ie 0.4ml:4ml. including
appropriate control tubes (donor only, recipient only)
4.. Spin down and resuspend in ~300ul of media and spread bugs onto at
35mm sterile millipore filter laid on a suitable agar plate and incubate for
5.. Remove the filter with flamed forceps and put it into ~10ml of media
(in a mccartney bottle or other suitable). Vortex thoroughly until all the
bugs are off the filter.
6.. Spin down the suspension discard the supernatant and resuspend in
suitable media (PBS if selection is to be on minimal media).
7.. Plate onto suitable selective media (this was the hard part for me)
at suitable dilution factors (try and see).
8.. grow overnight and good luck.
This method worked reasonable well, but I was told that shaking cultures is
a no-no for your donor as it shears off the sex-pilus
So I found this other method below which is easier (I thought so at least)
but smacks of Bucket Chemistry in that it is not so easy to reproduce, but
it seems to work better than the liquid culture method
a.. Solid Culture
1.. Grow confluent plates of donor and recipient bugs (1 plate of
donors, 4 plates of recipients).
2.. After overnight growth harvest 1/4 of the donor plate with a flamed
loop and place it in a pile on a fresh agar plate. Harvest all the bugs off
1 recipient plate and add them to the donors (a bit at a time you can only
scrape off so much at any one time)
3.. Once the harvesting is complete mix the pile of bacteria with the
loop until your happy that it is "well mixed" a bit arbitrary I know but mix
4.. Repeat for other plates and remaining donor . I usually end up with
4 piles on one Nutrient Agar plate.(Depends how many transconjugants you
want to get back, I wanted lots)
5.. Incubate for 4h @37C and harvest the piles into broth and spin down
and plate as for above method
As messy as it sounds the second method yielded much higher numbers of
transconjugants is a hell of a lot easier
Nick van Holst
Paul Beare wrote in message ...
>Does anyone have a reference for conjugation methods. I'm currently having
>problems getting transconjugants using the E.coli strain S-17 (donor) and
>the Pseudomonas aeruginosa strain PAo1 under selection for both
>tetracycline and kanamycin.
>Paul Beare B.S.c (Hons)
>Department of Biochemistry
>University of Otago
>PO Box 56 Dunedin
>Email: paul_b at sanger.otago.ac.nz
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