Prepare:

200ml LB in a 1L flask

1L sterile ddH2O and plAce in cold room

4 50ml conicals chilled on ICE

10% glycerol (cold)

Chilled boxes of 100ul and 1ml pipet tips.

Chilled 0.5ml tubes.

GENEral

This protocol is for generating enough Cells for 10-20 electoporations. Its primary advantage is that it avoids the use of any big and heavy rotors. It can be scaled up or down depending on your needs.

growth

1. Innoculate 2mls LB + antibiotic with a single colony. Its best if the colonies are not old (less than 2 weeks).

2. Innoculate 200ml of prewarmed LB + antibiotic with at least 1:50 dilution of the O/N culture. A 1:100 is my usual but it depends on the strain and temperature.

3. Grow cells at 37C to 0.5-0.8OD. I usually try to hit 0.7-0.8OD. Time varies greatly based on strain and temperature so can take as little as 3 hours or up to 7 hours. Check OD frequently after the first 2 hours.

Washes (Keep everything cold throughout procedure for best results)

4. Pour cells into the chilled 50ml conicals and leave on ice 60min (30min minimum). This step can be skipped but efficiency drops significantly for some reason.

5. Spin down cells 10min, 3K rpm, 4 C, pour off supernatant. The RT6000 with the swinging bucket rotor works great for his.

6. Add 20ml of cold ddH2O to each tube and vortex for 10 seconds. Bring all the tubes up to 50ml with ddH2O then spin to pellet.

7. Wash the same way 2 more times for a total of 3 washes.

8. Resuspend cells in each tube in 1ml (use the chilled tips) by quick vortex in cold sterile 10% glycerol.

9. Pellet as before, aspirate off glycerol (don't pour), and resuspend cells in each tube of cells in 100ul cold sterile 10% glycerol (chilled tips). The total volume should be 500-600ul.

10. Transfer 50ul to each chilled 0.5ml to using a chilled pipet tip and freeze on dry ice. Cells can be stored at -80C with little loss in effiency for about 6 months.