Electroporation transformation of E. coli

1. Preparations:

• Put a bottle of NZY+ at 37C.

• Put cuvettes (either individually wrapped new ones or the re-used and re-autoclaved ones that are in a rack, wrapped in foil), at 4C

• Label a 1.5ml microfuge tube for each transformation, including 0 DNA, keeping it sterile.

• Put one tube of electrocompetent cells per transformation on ice to thaw.

• When everything is cold/warm, put the plastic slide in the electroporator and put everything else, including pipettors and pipets, near the electroporator. Wipe the white parts of the pipettors with a little ethanol.

2. Check the electroporator settings:

• On/off switch is in back, to the left.

• Capacitance Extender 960

• Pulse controller 200 ohms

• Gene pulser 25 µFD

• For 0.1cm cuvettes (which we use): set Gene pulser to 1.6 kV

• For 0.2 cm cuvettes (Davis lab): set Gene pulser to 2.50 kV

3. Mix 1µl of DNA in a low ionic strength buffer, like TE, into a tube of 40µl of electrocompetent cells. Keep on ice.

4. Use sterile technique as much as possible. One at a time, transfer the 40µl of cells + DNA into a cold cuvette. Tap it on the table to get the cells to sit in the metal slot. Put the cuvette in the white plastic slide. Push it into the chamber until it stops.

5. Pulse by pressing continuously on the two red buttons that say "pulse", until it beeps.

• If it sparks or pops before beeping, release the buttons and go to step 6.

6. Immediately remove the cuvette, add 1ml NZY+ and pipet up and down carefully to get all the cells out. Transfer to a sterile, labeled microfuge tube.

7. Put the tubes at 37C for about one hour.

• Label one LB amp plate per transformation. Get the glass spreader and make sure there is ethanol in the beaker.

8. Spin 1 minute in the microfuge at setting 6. Check that there is a pellet and clear sup. Spin again if necessary.

9. Using a sterile pipet tip, remove the sup. Add 150 µl sterile water, pipet up and down to resuspend. Transfer to a selective LB plate (usually amp). Flame the glass spreader, let it cool for a few seconds and spread the cells.

• They are sticky, so it is hard to resuspend them.

10. When they have soaked in, turn them upside down and put at 37C. Check in 1.5-2 days.

• The amp breaks down, so don't let them sit in the incubator for more than 2 days. Make sure there are no colonies on the 0 DNA plate.