YEB medium recipe????

[Frederik Boernke]

Edgar Valencia wrote:
> 
> Hi Netters
> 
> I'm trying to prepare Agrobacterium tumefaciens competent cells and in the
> method I got the bacteria should be grown in "YEB" medium, the problem is
> the reference doesn't has the recipe and I couldn't find the original
> reference.
> Does anybody would share the recipe for this media?
> OTOH Does anybody has a protocol to prepare A tumefaciens competent cells
> or a electroporation protocol for them?
> Thanks a lot
> 
> Edgar

Hi,

here comes the medium

5g/l beef-extract (Difco)
1g/l yeast-extract
5g/l peptone (casein-hydrolysate)
5g/l sucrose
2 mM MgSO4

Competent Cells (An, 1987):

-inoculate 50 ml YEB with 2 ml of an o/n culture and incubate at 28 deg.
until OD600 is about 0.5 (ca. 4h)
- harvest cells via centrifugation at 5000 rpm/4 deg.
- resuspend pellet in 10 ml 0,15 M NaCl
- centrifuge again as above
- resuspend pellet in 1 ml icecold 75 mM CaCl2 sol.
- make 200 ul aliquots and freeze in liquid nitrogen
- cells can be stored at -80 deg for several months

Transformation

- thaw cells on ice
- add at least 15 ug plasmid-DNA and further incubate on ice for at 30
min while gently   mixing from time to time.
- freeze in liquid nitrogen and rapidly thaw at 37 deg.
- add 800 ul YEB and incubate 4 - 6 h at 28 deg
- plate aliquots on YEB agar plate with appropriate antibiotics
- incubate at 28 deg for 2 days

Alternatively you can prepare electrocompetent cells as described
for E. coli, works also quiete well.

If I can be of any further help, don't hesitate to contact me.

Cheers
Ricky

******************************************************************

Frederik Boernke
Research Group of  Molecular Plant Physiology
Institute for Plant Genetics and Crop Plant Research (IPK)
Corrensstr. 3
06466 Gatersleben
Tel.  039482 -5 321
Fax. 039482 -5 515
http://www.ipk-gatersleben.de