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now: Sun Apr 11 00:47:18 2021 ... mod: Sun Apr 4 18:44:13 2021
Electroporation-mediated Plasmid Transformation of Bacteria
Electroporation is the process whereby bacteria exposed to a strong, transient electric field develop holes in their cell wall and plasma membrane. Plasmid DNA in solution near these permeablized bacteria is then taken up into the bacterial cell. The bacteria spontaneously heal the holes after the treatment. This is one of the most efficient methods of transforming bacteria known with efficiencies of up to 1010
transformants per microgram of plasmid electroporated.
- thaw a 50μl tube of electrocompetent cells on ice
- add 5μl plasmid solution in water or TE to the bacteria and mix well
- it is crucially important that there be very little or no salt in the DNA solution. salt can be removed from plasmids by either EthanolPrecipitation or column chromatography (eg: GFX column) followed by resuspension in water or TE. the presence of salt in the electroporation will cause arcing of the electric field and the failure of the transformation
- remove 50 μl of the bacteria / plasmid suspension and add to a 0.1 cm x 1.0 cm electroporation cuvette that has been pre-chilled on ice
- electroporation cuvettes also come in 0.2 cm and 0.4 cm gap widths, which are used for electroporation of yeast or tissue culture cells. be careful not to get the wrong size
- by gentle tapping on a hard surface ensure that the bacterial suspension is well distributed at the bottom of the cuvette
- be very careful not to introduce any bubbles into the bacterial suspension in the cuvette. bubbles will also cause arcing of the electric field and failure of the transformation
- the bacteria in the cuvette can be stored on ice if desired
- set the electroporator to 1.8 kV (1800 V), remove the electroporation cell holder, wipe the cuvette containing the bacterial solution with a kimwipe and place in the holder, insert the holder into the electroporator and activate the field (by pressing the pulse button twice)
- if you hear a loud popping sound, the electroporation has arced and the transformation has failed. discard the cuvette and bacteria, remove salt from the plasmid DNA by EthanolPrecipitation or column chromatography and repeat the electroporation procedure.
- the electroporator can display the time constant of the electric field decay after electroporation. a value of 5.0 down to about 4.0 generally indicates success
- immediately add 1.0 ml prewarmed bacterial medium to the cuvette, pipette gently up and down to suspend the bacteria, transfer the bacteria to a 1.5 ml eppendorf tube and incubate at 37C for bacterial recovery and initial expression of transformed antibiotic resistance genes
- for ampicillin resistance, as little as 5 minutes at 37C will suffice, for all other antibiotic resistance genes, a minimum of 20 minutes at 37C before plating is recommended (also fine for ampicillin resistance)
- plate 50 μl of the bacterial suspension on a bacterial plate with suitable antibiotics
- this is the "low" concentration plate representing an approximately 50x dilution of bacteria from the "high" concentration plate described below. at least one of these two plates will generally give rise to suitably isolated bacterial colonies
- centrifuge the remaining ≃1 ml of suspension at 12000 g for 1 minute, gently pour off the supernatant, leaving around 120 μl of liquid on the bacterial pellet in the eppendorf tube, resuspend the pellet thoroughly in this liquid by pipetting up and down and plate the entirety of the resuspension on a second bacterial plate with appropriate antibiotics
- this is the "high" concentration plate
- incubate both plates overnight at 37C