Electroporation transformation of Agrobacterium cells
Agrobacterium strain
Antibiotic selection
C58C1
Gent, 50 μg/ mL (Gent resist gene in helper plasmid),
Rif 50 μg/ mL (for strain- Rif resist gene in genome)
EHA101
Kan 50 μg/ mL (Kan resist gene in helper plasmid),
Chlor (for strain- Chlor resist gene in genome)
LBA4404
Rif 50 μg/ mL (for strain- Rif resist gene in genome)
Plant transformation vector
Antibiotic selection
pZBL1N
Kan (50 μg/ mL) bacterial AND plant Kan (75 μg/mL) resist
genes
pZP211, pZP212
Strep/ Spec (100 μg/ mL for each) for bacteria, Kan (75 μg/ mL)
for plant
NOTES: Can use LB media for everything, but YEP has less salt and Agro grows better in this media. Can use
AB minimal media with sucrose for Agro because E. coli does not grow on sucrose.
1. Before you begin the transformation, make sure you have the appropriate selective plate(s), cuvette(s), sterile
pasteur pipet(s), sterile culture tube(s). Put plates at 28oC before beginning the transformation.
2. Set up electroporator (can do this while cells are thawing) = BioRad Gene Pulser.
Settings for the three components:
NOTE: different settings than for electroporation transformation of E. coli.
VERY IMPORTANT: make sure the leads in the front go from the Gene Pulser to the Pulse Controller and then a
second set of leads from the Pulse Controller to the Safety Chamber. The leads in the back go from the Gene
Pulser to the Capicitance Extender. There may also be a grey cable from the back of the Gene Pulser to the back of
the Capacitance Extender. This doesn=t seem necessary for E. coli electroporation since some setups I have used
don=t have it.
Gene PulserC CAP (capacitance) dial at 25.0 μFD
VoltsC press set volts button and then use Araise@ button to set at 1.8 kV
Pulse ControllerC 100 OHMS (Ω)
Capacitance ExtenderC 960 μFD (doesn=t actually get used when you set the Gene Pulser
to 25 μFD, so it really doesn=t matter, but this is the setting I use)
Also, make sure you locate the Safety Chamber sliding tray and that it is in a freezer.
3. If using frozen electroporation competent cells, then gently thaw them- put on ice IMMEDIATELY and don=t
thaw with hands. If using freshly prepared electroporation cells, then keep them cold the whole time.
4. Add 1 μg plant transformation vector to 50 μL of electroporation compenent Agrobacterium cells in a cold
Eppendorf tube.
5. Incubate on ice for 30 sec.
6. Transfer cells to a cold 0.2 cm electroporation cuvette and shake suspension to the bottom so that it covers the
whole bottom of the cuvette.
7. Wipe condensation from the metal plates of the cuvette quickly and thoroughly. Place cuvette in the Safety
Chamber sliding tray and push slide into the chamber until the metal plates are seated between the contacts.
8. Press and hold the two red pulse buttons simultaneously until the Gene Pulser beepsB usually about 5 - 10 sec.
This produces a time constant of __________ and the field strength will be ______________.
9. Remove the cuvette from the chamber and immediately add 1.0 mL YEP that is at RT.
10. Transfer sample to a culture tube and put into incubator shaker at 28oC for 1 hr shaking at around 220 rpm.
11. Plate all onto selective media plates. Because 1 mL is too much to add to a plate, spin the cells into a pellet by
pulsing in a microcentrifuge (you don=t want the g forces on the cells to get too high for too longB the cells are
quite fragile at this point).
12. Incubate inverted plate(s) at 28oC until colonies appear. Usually takes 2 daysB Agro. grows slower than E.
coli.
13. As always with bacteria, wrap plates with parafilm and store inverted at 4oC.
YEP media (per L):
Difco Bacto-peptone
Difco Bacto yeast-extract
NaCl
Difco Bacto Agar
10g
5g
5g
12g
pH to 7 with NaOH (may not require adjustment)
dmr 081302 (from Amanda Walmsley)