Electro competent cells
From HuLab Wiki
Preparation of Electro competent E. coli cells (per the BioRad directions)
1. Inoculate 1 liter of 2XYT with 1/100 volume of a fresh overnight culture. (Leo grows in a 1L Fernbach flask)
2. Grow cells at 37°C with vigorous shaking to an OD600 of approximately 0.5-0.7 (the best results are obtained with cells that are harvested at early- to mid-log phase; the appropriate cell density therefore depends on the strain and growth conditions.
3. To harvest, centrifuge cells in cold centrifuge bottles in a cold rotor at 4000xg for 15 minutes.
4. Remove as much of the supernatant as possible. Sacrifice the yield by pouring off a few cells rather than leaving any supernatant behind.
5. Gently resuspend the pellets in a total of 1 liter of ice-cold 10% glycerol taking care not to lyse them. Centrifuge as in step 3.
6. Resuspend in 0.5 liter of ice-cold 10% glycerol. Centrifuge as in step 3.
7. Resuspend in 250 ml of ice-cold 10% glycerol. Centrifuge as in step3.
8. Resuspend to a final volume of 3 to 4 ml of ice-cold 10% glycerol. The cell concentration should be 1-3 x 1010 cells/ml.
9. Freeze in aliquots on dry ice or liquid nitrogen and store at -70°C.
Do not autoclave or filter-sterilize the glycerol; prepare fresh weekly
2L of 10% glycerol: make fresh each time
200ml glycerol
1800 ml of mqH20
