Difference between revisions of "Team:Birkbeck/Electroporation"
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Revision as of 17:12, 17 September 2015
Transformation of plasmids into electrocompetent cells
Electroporation protocol.
- 1. Thaw out the 50 µL cell suspension aliquot on ice for ca. 30 minutes. Concurrently, cool electroporation tubes on ice.
- 2. Add 100pg-1µg of plasmid DNA to the cell suspension (approximately 1-5 µL of plasmid solution).
- 3. Mix plasmid and cells thoroughly and continue to incubate on ice for 5-10 minutes.
- 4. Transfer the mixture of cells and plasmids to an electroporation tube.
- 5. Electroporate cells; for E.coli, this is done at 2.5 kV, 200 Ohms, 25 uF.
- 6. Immediately add 450-950 uL of LB broth.
- 7. Incubate tubes at 37oC for 60-90 minutes in a statis incubator.
- 8. Plate 100 uL of cells onto agar plates with appropriate antibiotic resistance.
- 9. Incubate overnnight at 37oC in a static incubator.
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