Method prepared by Grace Panganiban: gepangan@facstaff.wisc.edu


Electroporating (transforming) bacteria


(We will be using trp- bacteria strain MC1066 and a BioRad Gene Pulser)

1) Place cuvettes on ice, add 2 ul of DNA to each.

2) Get cuvette holder (from Jeff's drawer), 2xYT, pipettemen, and tips ready by the Culbertson lab (4th floor) electroporator.

3) Turn on electroporator (switch on back). Set voltage to 2.5 (max), and capacitance to 25.

4) Thaw electrocompetent MC1066 cells (stored at -80oC) on ice. You will need 40 ul for each sample. They are in either 500 ul or 1000 ul aliquots. (Make sure everything else is ready before you thaw the cells)

5) Mix cells to generate a uniform suspension, then add 40 ul to each cuvette. Make sure the cells contact the DNA, and that there are no bubbles in the cuvette.

6) Put the cuvette in the cuvette holder, slide it into the chamber such that the metal of the cuvette contacts the electrodes.

7) Depress both red buttons, and hold them until the machine beeps*.

8) Put the cuvette on ice, and add 1 ml of 2xYT immediately.

9) After electroporating all the samples, transfer the bacteria and the 2xYT to clean, labelled eppie tubes, and incubate at 37oC for 1 hr.

10) While the bacteria are recovering, put an appropriate number of B min/amp plates in the 37oC incubator to prewarm.

11) Following the 1 hr recovery, plate 200 ul from each sample onto prewarmed and labelled B-min/amp plates.

12) Incubate overnight at 37oC.

13) Store plates at 4oC till ready to set up minipreps.

* If you have arcing (i.e. the electroporater short-circuits), there is too much salt in your DNA prep. Repeat the transformation with 0.5 ul of DNA and a clean cuvette.

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