Chris:LabNotes/General Protocols/Electroporation
Jump to navigation
Jump to search
E. coli Electroporation Protocol[edit]
Background[edit]
- Instead of chemical transformation, there are circumstances in which one may want to use higher efficiency electroporation for bacterial transformation of plasmids
- You will need:
- Electroporation machine - in Palsson lab (in cell culture room on desk)
- Cuvettes - Gene Pulser Cuvette, E. coli Pulser Cuvette (Bio-Rad, Catalog No. 165-2089)
- SOC media (want to be equilibrated to RT at time of use)
General Notes[edit]
- Prior to electroporation, everything must be kept on ice in order to ensure that it is cold. Consequently, have cells, DNA, and cuvettes on ice prior to use.
- Because electroporation is so efficient with bacteria, only need femtogram levels of plasmid in order to have good transformation (may need to do a few titrations prior to using plasmid)
Protocol[edit]
- On ice, we want to add 50-60ul of cells + small volume of DNA plasmids (max 5ul, fg levels)
- Turn on machine and set volts to 1800 (based on specification of cuvettes)
- Take off the cap of the cuvette and discard. Pipette in samples into the middle of metal plates/slot within cuvette (will not be able to reach all the way down with pipette)
- Tap the cuvette to make sure cells go to the bottom in between the two metal plates/slot and keep on ice
- Take cuvette out of ice and wipe with kimwipe to remove precipitation on sides
- Put cuvette in plastic holder (will have a notch on one side of cuvette so there is only one way to place into holder)
- Push holder into slot in machine such that the metal sides of the cuvette make contact with metal plates in machine
- Press "Pulse" twice and wait for beep
- Immediately take out cuvette from holder and add 250ul SOC and pipette up and down. It is essential to do this immediately after pulse/beep in order to save the bacteria
- Use p200 pipette to transfer as much of the cells from cuvette into an Eppendorf tube (can tilt cuvette slightly to optimize volume of cells recovered)
- Incubate at 37C for a few hours and plate or grow in liquid media with antibiotic overnight
- Discard cuvette after use