Preparation of Electroporation-Competent E. coli Cells

1. Streak one loopful of E. coli, XL1-Blue or SURE cells, on an YT or YT-Tet20 agar plate (20 ug Tetracycline / ml of YT agar).

2. Inoculate 3 ml of YENB (Add to 1 liter of distilled water, 7.5 grams of Yeast Extract [0.75%], and 8 grams of Nutrient Broth [0.8%]; autoclave.), and grow culture overnight in a shaking incubator [250rpm] at 37 degrees C.

3. Inoculate the 3 ml overnight culture into 1 liter of YENB, and grow to an OD600 of 0.5 – 0.9. This will typically require approximately 3-4 hours in a shaking incubator [250rpm] at 37 degrees C. Should the cells be overgrown, dilute to an OD600 of 0.2, and re-grow to an OD600 of 0.5 – 0.9.

4. Harvest the cells. Chill the flask on ice for approximately 5 minutes. Transfer the cells into sterile Nunc 200 ml centrifuge bottles (Fisher Sci cat# 14-962-171; Nunc # 376813), and centrifuge at 2000 x g for 5 minutes at 4 degrees C (Sorvall Legend Mach 1.6 R with swinging bucket rotor). (The viability of the culture and, thus, the electrotransformation efficiency, will decrease with increases in centrifugal force and length of spin.)

Note: Remaining steps should be performed in a cold room if available. Approximately ~600 ml of sterile, ice cold 1mM Hepes, and 150 ml of sterile, ice cold 10% glycerol (prepared with 1mM Hepes buffer) will be required.

5. Remove and discard medium. As the culture medium does not contain added salts, it is not important to remove traces of medium. Resuspend each pellet in 10 ml of ice cold 1mM Hepes. Decrease the number of centrifuge bottles by 50% until only two bottles remain. Add 130 ml of cold 1mM Hepes per bottle. Centrifuge at 2000 x g for 5 minutes at 4 degrees C; decant. Repeat wash steps two additional times using 150 ml of cold 1mM Hepes. After the final wash step remove all traces of buffer using a pipet, but be careful not aspirate cells.

6. Resuspend the cells (from 1 L of culture) in 2 ml of 10% ice cold sterile glycerol in 1 mM Hepes to give salt-free, concentrated electrocompetent cells.

8. Aliquote 120 ul of these electrocompetent cells into 1.7 ml microcentrifuge tubes and immediately freeze by placing in a crushed dry ice / ethanol bath and then store at -80 degrees C until needed.