Agrobacterium Protocols
Protocol adapted from Wise, Liu et al. 2006.
Agrobacterium Media
Yeast Extract Beef (YEB) medium (pH 7.2)
5 g/L - tryptone
1 g/L - yeast extract
5 g/L - nutrient broth (or 2.5g/L of L. B. Medium or 5g/L peptone)
5 g/L - sucrose
0.49 g/L - MgSO4·7H2O
LB medium (pH 7)
10 g/L - tryptone
10 g/L - NaCl
5 g/L - yeast extract.
(Add 15 g/L agar for solid medium)
Supplies and Solutions for Agrobacterium Transformation
Freeze/Thaw Method
20 mM CaCl2 (autoclaved).
1M CaCl2 Stock solution (14.7g 100ml H2O)
20mM CaCl2 (1ml 1M CaCl2 Stock 50ml H2O)
Liquid N2
Electroporation Method
10% Glycerol (autoclaved).
Electropulse generator (Bio-Rad, Gene Pulser II).
0.2-cm Electroporation cuvette (Bio-Rad, cat. no. 1652086).
Transformation of Agrobacterium
Using the Freeze/Thaw Method
Preparation of Freeze/Thaw Competent Agrobacterium Cells (F/T CC)
Inoculate Agrobacterium strain in 2mL of liquid growth medium (YEB). Include needed antibiotic(s) (Rifampicin). Grow overnight with shaking at 25 to 28°C (so the plasmid in the strain is retained).
Use the 2mL culture to inoculate 50 mL of the same medium in a 250mL flask. Incubation until OD600 between 0.5 and 1.0 (Grow at 18°C for maximum transformation efficiency but will take 2.5 to 3 days). Dilute and grow with YEB if OD is exceeding.
Chill the culture on ice. Pellet the cells by centrifuging at 4°C for 10 min at 10,000g in 50ml falcon tubes. Discard the supernatant. Resuspend cells in 5 mL cold 20 mM CaCl2 and repeat centrifugation. Discard the supernatant (wash step needed to remove antibiotic(s)).
Resuspend the cells in 1 mL cold 20 mM CaCl2. Aliquot 50-100 µL of the cell solution into the chilled Eppendorf tubes, freeze in liquid N2. Store extra tubes of cells at –80°C for future use. Fresh cells can be used before freezing for transformation.
Summary: 2ml YEB + Rif 50ml YEB+ Rif OD > 0.5 & < 1 wash with 20 mM CaCl2 twice 1ml 20 mM CaCl2 split to 50 µL -80°C
Transformation through Freeze/Thaw Method
Use plasmid DNA at a concentration of 0.1 to 1 µg per µL.
Place frozen competent cells from freezer on ice. Add 0.1-1 µg plasmid DNA to cells before thawing (up to 10 µg can be used).
Freeze the mix by lowering the tube into liquid nitrogen for about 5 min (Dry ice and ethanol can also be used but with lesser transformation).
Thaw the frozen mixture for 5 to 10 min at room temperature. Add 1 mL of growth medium (YEB) and incubate at 25–28°C for 2 to 4 h.
Pellet the cells by spinning for 1 min at high speed in a microcentrifuge. Discard 800-900 µL of the supernatant and plate 100 to 300 µL cells on an agar plate (LB) containing antibiotic(s) appropriate for selection of transformants (GV3101 will take 2 days).
Summary: F/T CC + plasmid DNA 0.1-1 µg freeze Thaw add 1ml YEB incubate 2-4 h spin and reduce vol to <300 µL plate on LB plates with antibiotic(s) for selection.
Electroporation method
Preparation of Electro-Competent Agrobacterium Cells (ECC)
Inoculate Agrobacterium strain in 2mL of liquid growth medium (YEB). Include needed antibiotic (Rifampicin). Grow overnight with shaking at 25 to 28°C (so the plasmid in the strain is retained).
Use the 2mL culture to inoculate 50 mL of the same medium in a 250mL flask. Incubation until OD600 between 0.5 and 1.0 (Grow at 18°C for maximum transformation efficiency but will take 2.5 to 3 days). Dilute and grow with YEB if OD is exceeding.
Chill the culture on ice. Pellet the cells by centrifuging at 4°C for 10 min at 10,000g in 50ml falcon tubes. Discard the supernatant. Wash cells with 20 mL ice-cold sterile water 2 times. Discard the supernatant (wash step needed to remove antibiotic(s)).
Finally wash using 10 mL ice-cold 10% glycerol instead of water. Resuspended in 1 mL cold 10% glycerol (10% glycerol can be used for all washes).
Aliquot 50 µL of the cell solution into the chilled Eppendorf tubes, freeze in liquid N2. Store extra tubes of cells at –80°C for future use. Fresh cells can be used before freezing for transformation.
Summary: 2ml YEB + Rif 50ml YEB+ Rif OD > 0.5 & < 1 wash with 20 mL cold water twice wash with 10 mL cold 10% glycerol and resuspend in 1 mL 10% glycerol split to 50 µL -80°C
Preparation of Electro-Competent Agrobacterium Cells (ECC)
Prepare for the electroporation process by first programing the electroporation apparatus
Electricity - 2400/ 2500 V
Capacitance - 25 µF
Resistance - 200/ 400 Ω
Distance between electrodes - 1/ 2 mm
(Note: If the apparatus has an inbuilt program for Agrobacterium use it as it will be optimized for its electroporation cuvet and the apparatus else standardize it using the value range given above)
Add 1-5 µL of plasmid DNA to the ECC and thaw them gently on ice. Mix gently and transfer cell/DNA mix to the chilled cuve (see to that the mixture touches both electrodes to complete the circuit). Fit the electroporation cuvet snugly into the holder.
Press the pulse button to deliver the electric pulse. Check “time constant” value displayed after pulse (7-8 ms give or take).
(Note: The time constant depends on the instrument and it tells us how fast the electric pulse moves through the cuvet, if it is too fast the arcing of the electric current occurs resulting in a loud popping sound. This is usually an indication that the cell or DNA solution contains salt. Arcing dramatically increases cell death and severely lessens the likelihood of obtaining viable transformants).
On successful pulse delivery without arching, immediately add 1 mL of the growth medium (YEB) to the cuvet and transfer the entire content to a 1.5 mL tube and incubate at 25–28°C for 2 to 4 h (in sterile conditions).
Pellet the cells by spinning for 1 min at high speed in a microcentrifuge. Discard 800-900 µL of the supernatant and plate 100 to 300 µL cells on an agar plate (LB) containing antibiotic(s) appropriate for selection of transformants (GV3101 will take 2 days).
Summary: Program the instrument add ECC+DNA to cuvet apply pulse on successful pulse add 1 mL YEB incubate 2-4 h spin and reduce vol to <300 µL plate on LB plates with antibiotic(s) for selection.
References
Wise, A. A., et al. (2006). Three methods for the introduction of foreign DNA into Agrobacterium. Agrobacterium protocols, Springer: 43-54.