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


Instructions for making electrocompetent bacteria


(We will be using trp- bacteria strain MC1066)

Day 1
1) Inoculate 4 mls of 2xYT (or LB) with a fresh bacterial colony.

2) Grow overnight at 37oC.

3) Place 1 liter of sterile deionized water; 500 mls of sterile 15% glycerol, and centrifuge rotors at 4oC to chill.

Day 2
4) Inoculate 400 mls of 2xYT (or LB) with the overnight culture.

5) Grow at 37oC till A600 ~ 0.5 (~2 hrs).

6) Meanwhile, fill several ice buckets, and use them to prechill centrifuge bottles, sterile deionized water (diw), and sterile 15% glycerol. Also fill 1 ice bucket with dry ice, and place a rotor in the centrifuge.

7) When bacteria have reached the appropriate optical density, chill the flasks for 15 min on ice, then pour the bacteria into chilled centrifuge bottles. From this point on, it is essential to keep the cells and everything that comes in contact with them ice cold.

8) Spin at 5000 rpm for 5 min to pellet the bacteria.

9) Pour off the medium.

10) Resuspend the cells in 200 mls of cold diw.

11) Spin at 5000 rpm for 5 min to pellet the bacteria.

12) Pour off the diw.

13) Repeat steps 10-12 2 times, making sure to keep everything cold.

14) Repeat steps 10-12 using 15% glycerol instead of diw.

15) Resuspend the bacteria in 2 mls of 15% glycerol.

16) Freeze bacteria in 500 ul aliquots in dry ice/ethanol bath.

17) Store at -80oC till ready to use for transformations.