YEB medium recipe????

[Edgar Valencia]

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

[John R. McQuiston]

It may be the YENB media, that is a salt free media for electroporation. I use
it with Brucella, Salmonella and E.coli and it works great. Since Brucella and
A. tumefaciens are very close genetically it'll probably work. It's in a
Biotechniques article for Salt free electrocompetent cells.
It's .75% Yeast Extract, .8% Nutrient Broth per liter I think.

You grow them to specific density, spin to pellet, wash once with sterile cold
water, spin again and electroporate. If you get on Biotechniques web site you
can find it.

John R. McQuiston

In article <Pine.SOL.3.95.981130114601.404A-100000@uxmal>, ed...@CIFN.UNAM.MX
says...

[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