Protocol 1: Electroporation of Agrobacterium tumefaciens with a plasmid of interest
Sarah M Prostak, Edgar M Medina, Erik Kalinka, Lillian Fritz-Laylin
Abstract
Electroporation is a widespread method of transforming competent Agrobacterium tumefaciens ( Agro ) cells with a plasmid containing a T-DNA of interest. The resulting Agro can be used to transform various plants and fungi, resulting in transformed cell lines. This protocol outlines the standard electroporation protocol we use to transform Agro in preparation for Agrobacterium -mediated transformation of the chytrid fungus Spizellomyces punctatus .
Before start
Electroporation should occur at least 4 days prior to the intended Spizellomyces transformation time to ensure that active, single colonies are available to be selected and transferred to liquid culture the night before transformation day (see Protocol "Growing liquid cultures of Agrobacterium prior to transformation day").
Attachments
Steps
Steps
Cool the following materials On ice at least 0h 20m 0s prior to starting:
- Plate with a lawn of wild-type Agrobacterium grown overnight at
28°C. - Purified plasmid(s) of interest.
- Molecular biology grade water, sterile.
- 10% (v/v) glycerol, sterile.
- 1.5 mL centrifuge tube(s), enough to hold the volume of Agro harvested.
- 0.5 mL centrifuge tube(s), one per plasmid to be transformed, plus controls.
- 2 mm electroporation cuvettes, one per plasmid to be transformed, plus controls.
Add 1mL to 2mL of ice-cold water to the plate of Agrobacterium . Hold the plate at ~45 degrees and run the water over the surface at least 3 times, gently scraping along the agar if necessary to recover the lawn of bacteria.
Transfer the 1mL of harvested cells to a 1.5 mL centrifuge tube, immediately place back On ice.
Pellet the cells at 4000rcf in a rotor prechilled to 4°C.
Remove the supernatant and gently resuspend the cells in 1mL of water. Do not vortex.
Repeat steps 4 and 5, 2 more times for a total of 3 washes.
Remove water and resuspend the cells in 800µL of cold 10% (v/v) glycerol, place tubes back On ice.
Add 50µL of cells to new 0.5 mL tubes, place back On ice.
Add 1µL of the plasmid of interest (200 to 300; directly from a miniprep should work) to its appropriate tube of cells, place back On ice and mix gently by pipetting .
Transfer the cells to cold 2 mm electroporation cuvettes, place back On ice.
Prepare the recovery media in advance by placing 150µL of Room temperature SOC medium to one 15 mL culture tube for each electroporation.
Turn on the electroporator and create an electroporation program with the following settings:
- Voltage= 2400V
- Capacitance = 25 μF
- Resistance = 200 Ω
- Cuvette size = 2 mm
Fully dry the cuvette before placing it into the electroporator chamber.
Electroporate your cuvette.
As quickly but as gently as possible, remove a little less than 150µL of SOC medium from the appropriate tube for the plasmid and pipette it into the cuvette. Remove the full volume from the cuvette and return it to the original culture tube.
Incubate culture tubes at 28°C, shaking at 225rpm.
Add 4-6 sterile glass beads to each LB plate.
Add 10µL cells to the appropriate plates.
Seal and invert the plates and incubate them at 28°C for about 4 days.
