competent agrobacteria - (Aug/28/2007 )
Some time ago I bought electrocompetent agrobacteria strain LBA4404.
Unfortunately, my attempt to electroporate the bugs weren't successfull and now I've just a couple of aliquotes of bacteria left.
I would like to establish a stock culture to keep at -80°C for further experiments.
I know that if I propagate them to establish a stock culture, agrobacteria will loose electrocompetence.
Moreover I am not sure about resistance to antibiotics to exploit in order to select bacteria. I checked manufacturers description (as vague as it can be) with database information and I'm a bit confused .
What are your general advices on this topic?
Can you suggest me a reliable protocol to obtain electrocompetent bacteria?
Thank you very much indeed!
I've grown agrobacterium EHA 105 (this is years ago) and was succesful in electroporating them (5msec, 12,5 kV/cm). The method used to make them electrocompetent is exactly as described in Sambrook's Molecular cloning: a laboratory manual from 1989 and electroporation with plasmid derived from e. coli (miniprep) was succesful from the first time. I think the antibiotic usage was pretty much as with E. coli, but not 100% sure on this one. I moved to a new lab since then...
Have you tried electoporating (with/without DNA) and plating on plates not containing antibiotics?
I'm going to follow Maniatis protocol.
My goal was to insert a certain plasmid in the Agrobacterium, but - apparently - I'm facing some kind of problem with the plasmid. In fact, none of my transformation experiments was successful, neither to clone plasmid in E. coli or to directly electroporate Agrobacterium, while controls were good in both cases.
After some attempts, I'm of course running out of plasmid and agrobacteria.
I could purify plasmid making a miniprep of a different strain containing the same construct.
Now I need "empty" agrobacteria to try electroporation again...
When I'll have obtained my Agrobacterium competent cultures, I will adopt your suggestion and add a bacteria control including an electroporation reaction without DNA.
Thanks for your help!