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Agro. Electroporation

Tonkyn, John C tonkyn at OCELOT.RUTGERS.EDU
Mon Nov 7 10:25:45 EST 1994


To:  Arabidopsis Community

Many thanks to everyone who sent protocols for preparing electrocompetent
Agrobacterium.  The protocols are the same or very similar to preparing
electrocompetent E. coli.  Here are some references followed by a few
protocols.  Thanks again!
John


We routinely electroporate Agrobacterium strains using the protocol by
Cangelosi, et al. in Methods in Enzymology 204:384-397.

Sharon Lafferty Doty
E.W. Nester's Lab


We use a protocol described in Mersereau et al. (1990) Gene 90:149-151 to 
prepare electroporation-competent agro.  We find that this methods works very
well.


Here are three :

1.	W. J. Shen, B. Forde, Nucleic Acids Research 17,  (1989).


2.	D. Mattanovich, et al., Nucleic Acids Research 17,  (1989).


3.	M. Mersereau, G. J. Pazour, A. Das, Gene 90, 149-51 (1990).


Brian Osborne
bosborne at nature.berkeley.edu


Agro electroporation protocols are:
Mattanovich NAR 17(16) 6747'1989
Wen-jun NAR 17(20) 8385'1989
For routine things I use freeze/heat shock protocol which is real simple 
but less efficient. Can provide if needed.
Regards
Dima Belostotsky
U of Georgia
Dimtry at bscr.uga.edu


We find that the method in Nagel et al (1990) FEMS Microbiol. Letts 67,
325-328 works well for A.t. (pGV3850).

Andy
Andy.Maule at BBSRC.AC.UK


Dear John Tonkyn,

We routinely electroporate Agrobacterium strains using the protocol by
Cangelosi, et al. in Methods in Enzymology 204:384-397.

Sharon Lafferty Doty
E.W. Nester's Lab


I prepare electrocompetent Agro just as I do with E. coli (see Current
Protocols in Molecular Biology), except culturing them at 28 degrees.  I
get an efficiency about 1E6 transformants/1 ug of pBI121, using GV3101.

Good luck,

Fumi Katagiri
Ausubel lab
Dept. of Molecular Biology
Mass. General Hospital


We have not tried electroporation yet, but Biorad maintains an up-to 
date biblio of transformation papers by species. Their Tech line could 
help.  I do not have a current list.  The old list cites NAR 17, 6747 
1989.  I would look at the Rhizobium papers too as the two are related.

Rick Amasino                         phone: 608-262-4704
Dept Biochemistry                    Fax: 608-262-3453
420 Henry Mall                       email: amasino at biochem.wisc.edu
University of Wisconsin
Madison WI 53706-1569



I use a modification of a protocol from the Ausubel et al "current 
protocols" which is for E coli. For agro:
1. inoculate 1 liter of L broth with 1/100 volume of a fresh overnight

2. grow cell to OD600 of 0.5-1.0

3. Harvest cells:  chill flask on ice 15-30 min, centrifuge in a cold
rotoor at 4000xgmax for 15 min

4. remove as much of the sup as possibe.  resuspend pellets in a total
of 1 liter of cold water. c'fuge as in step 3.

5. resuspend in 0.5 liter of cold water, cfuge as in step 3.

6. resuspend in 20 ml cold 10% glycerol.  cfuge as in step 3.

7. resuspend to a final volume of 2 to 3 ml in cold 10% glycerol.
The cell concentration should be about 1-3 x 10exp10 cells/ml

8. this suspension may be frozen in aliquots on dry ice and store
at -70C.  (80 ul aliquots) cells are good for at least 6 months.

Electrotransformation:

1. gently thaw cells at room temp and place them on ice.

2. In a cold 1.5 ml polyprop tube, mix 40 ul of suspension with 1-2 ul of
DNA (DNA should be in a low ionic strength buffer such as TE) Misx well 
let sit on ice 0.5-1 min. (I use 1 ul of a 1:20 dilution of a standard
miniprep of DNA)

3. set the gene pulser apparatus at 25 uF and 2.5 kV.Set the pulse 
controller to 400 omega.

4. transfer the mixture of cells and DNA to a cold, 0.2 cm electroporation
cuvette and shake the suspension to the bottom of the cuvette.  Place the
cuvette in a chilled safety chamber slide.  Push the slide into the chamber
until the cuvette is seated between the contacts in the base of the 
chamber.

5. pulse once at the above settings. this should produce a pulse with a
time constant of 10 msec (check this, I'm not sure...)

6. remove cuvette from the chamber and immediately add 1 ml of SOC
medium to the cuvette and quickly resuspend the cells with a pasteur
pipette.
(this rapid addition of SOC after the pulse is very important in maximizing
the recovery of transformants.

7, transfer the cell suspension to a 17 x 100 mm polypropylene tube and
incubate at 28 C for 1 hour.

8. plate on selective media.

2 days later you should see colonies..

L broth: 1% bacto tryptone, 0.5% Bactoyest, 0.5% NaCl

SOC:  2% Bacto tryptone, 0.5% Bact Yeast extract, 10 mM NaCl, 2.5 mM Kcl
10 mM MgCl2, 10 mM MgSO4, 20 mM glucose.

good luck, jean greenberg



I have used the following to successfully make competent
LBA4404.

Grow a seed culture of LBA4404 to saturation (36-48
hours).  You will need about 5 ml.

Late in the day before you want to make the competent
cells, inoculate 1 liter of YEP media 1:200 with the
saturated agro culture.

Grow the agro culture to OD (600 nm) of 1.5.

Proceed on ice.

Harvest cells by centrifugation, 5K RPM, 15 min.

Resuspend bacteria in 1 liter cold sterile dd water.

Centrifuge 5K RPM, 15 min.

Resuspend bacteria in 500 ml cold sterile dd water.

Centrifuge 5K RPM, 15 min.

Resuspend bacteria in 20 ml cold, sterile 10% glycerol.

Centrifuge 15 min at 6K RPM.

Pour off supernatant.

Add 1 ml 10% glycerol (total is now about 2 ml).

Make 80 ul aliquots (the suspension will be viscous).

Freeze on DRY ICE and store at -80 C.

Use for electroporation as per your instrument.  Very
little DNA is needed (on the order of 0.5-1.0 ul of
miniprep DNA is plenty).

Immediately add 1 ml media to the cells (YEP or LB
without selection).  Grow at 28 C with shaking for 2-4
hours.  Plate 10 and 100 ul on selective media.


YEP Media (1 liter)

10 g Bacto Peptone
10 g Yeast Extract
5 g NaCl


Best of luck

Michael Sullivan
MSU-DOE Plant Research Lab
Michigan State University



Hi, John, 

You can find the original protocol in: Cangelosi et al. 1991. Methods in 
Enzymology vol. 204: 384--397. There is also a much simplified method 
described by Charles and Nester in J. Bacteriol. 1993. vol. 175(20): 
6614-6625.  Briefly, you scrape agrobacterial cells from an agar plate, 
wash them three to four times with distilled water and suspend them in 
minimal volume of sterilized distilled water. And you are ready to go! 
If your purpose in to introduce plasmids, this simplified procedure works 
just fine. The trick is to avoid salts in your DNA preps and add as much 
DNA as possible. For some Octopine type Agrobacterium strains, the 
efficiency is low for some unknown reasons. You can increase your chance 
greatly by using a lot of Agro cells.  Good luck!



Hi John,
I routinely have electoporated plasmids in Agrobacteria strains (LBA4404 and 
EHA101) using an adaptation of the method described by Dower et al, 1988
NAR 16: 6127-6145 in which E.coli electrporation is described.
The procedure I used is outlined below:

A single Agrobacterium  colony is used to inoculate 5 ml of LB + appropriate 
antibiotics. This culture was grown for 48 hours at 28C in a shaking incubator.
This 5 ml culture was used to inoculate 500 ml of LB + appropriate antibiotics.
This culture was grown at 28C until the culture reached a density of
OD600 = 0.5 (approx. 4 hours).  The cells were spun down (10 min, 5000 x g) 
and resuspended in 500 ml of sterile H2O (repeated 2x).  The cells were spun
again and resuspended in 3 ml sterile H2O,  containing 10% glycerol.  40 ul
of the cells were aliquoted in eppendorf tubes and either used directly
for electroporation, or stored at -80C for  future use.

For electroporation I use a Bio-Rad gene-pulser with 0.2 cm cuvettes. 
The pulse generator is set to 25 uF capacitor, 2.5 kV and 200 ohm in parallel 
with the sample chamber.

After electroporation the cells are grown for 2 hours at 28Cin a shaking 
incubator before plating (10-100ul) on LB plates containing  appropriate
antibiotics. Typical transformation frequences are 10E7 -10E8 colonies/ug DNA.

I hope this helps,

Gijs
vanrooijen at abrsle.agr.ca
Wanyin Deng (Nester Lab)
Univ. of Washington, Seattle



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