bacterial transformation

Ramanathan Vairamani rv21 at cornell.edu
Thu Aug 27 10:35:44 EST 1998


Hey,

Why not do a triparental mating?  You have two E. coli strains - one with
your binary and the other with a helper plasmid like pRK2013.  And the
third parent is your Agrobacterium strain.  Mix toothpick scoops of these
three parents (from plates) on a plate.  Let the mix have fun for about 10
h.  Resuspend the mat in a diluent, serially dilute and plate and you will
have your transconjugant Agro within the next few days. You have to use AB
minimal medium with the apt antibiotics for selecting the
transconjugants.  AB minimal will counterselect E. coli.

Send me an e-mail if you want more info.

Ram
rv21 at cornell.edu


In article <35E46BF3.57C2 at bio1.lan.mcgill.ca>, Mona Damaj
<mdamaj at bio1.lan.mcgill.ca> wrote:

> Hello,
> 
> We are facing problems in tranforming Acetobacterium (a gram-positive
> bacterium). We tried the electroporation of the cells, but it did not
> work. We then tried to remove the cell wall by lysozyme digestion before
> electroporation, but it did not work. We are now thinking of partially
> digesting the cell wall.
> 
> Any helpful suggestions or protocols!
> 
> Thanks.



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