- Grow 6 ml of bacteria overnight
- On the next day harvest cells at 16,000 X g for 1-2 min at R.T.
- Wash twice the cell pellet with 1 ml of 300 mM sucrose at R.T.
- Ressuspend the pellet in 100 µl of 300 mM sucrose.
- Electroporation is made in a prechilled 2 mm gap width electroporation cuvette.
- Mix 100 ng of plasmidial DNA with 100 µl of eletrocompetent cells.
- MicroPulser Electroporator BioRAD sets: 25 µF; 200 Ω; 2.5 kV
- Immediately following electroporation, 1 ml of SOC (2% tryptone, 0.5% yeast extract, 10 mM NaCl , 10 mM MgSO4, 10 mM MgCl2) is added.
- Cells are then incubated at 37°C for 2h to allow expression of the AbR phenotype.
- Seed the bacteria on appropriate plates and incubate O.N. at 37°C.
Reference:
Choi K-H; Kumar A; Schweizer H.P. A 10-min method for preparation of highly eletrocompetent Pseudomonas aeruginosa cells: Application for DNA fragment transfer between chromosomes and plasmid transformation. J. Microbiol. Methds. vol. 64:391-397, 2005