Competent Agrobacterium protocol?

Michael J. Prigge prigge at darkwing.uoregon.edu
Mon Jun 2 04:00:10 EST 1997


In article <3391f56c.105668614 at news.iastate.edu>, ckafer at iastate.edu (Chris
Kafer) wrote:

> The electrocompetent protocol I have requires 7-8 washes which is a
> bit time consuming.  I was wondering if anybody out there had a
> chemical competency protocol akin to the TSS or Inoue method for these
> bugs.

I have a protocol that I used once successfully. Hopefully I'll be using it
alot in the near future. I don't have a ref, but I think it came from Steve
Kay's lab. 

Grow 1 L agro w/ app. antibiotics to OD550 of 0.5 to 1.5
Chill on ice
Spin at 4C, discard sup
Resuspend in 20 mL of cold, sterile 20 mM CaCl2
Aliquot & freeze in liquid N2 (200 ul/tube, I think I used)
Store at -80C

Then...
Add 1 ug supercoiled plasmid to tube
Incubate in 37C H2O bath for 5 min
Freeze in liquid N2
Incubate at 37C again for 5 min
Add 1 mL LB
Leave on benchtop for 2 hrs
Spread 200 ul on appropriate plate
Michael Prigge                          Phone: (541) 346-4256
Institute of Molecular Biology            Fax: (541) 346-5891
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