BactoZap Competent Cells

#LB10:5:5 (1 L)
10 g of Tryptone or Peptone
5 g of Yeast extract
5 g of NaCl

#15% Glycerol

1. Prepare 100 ml of sterile LB10:5:5 + 1 ml Tritech Research CompBooster solution in a 500ml flask.
2. Grow cells for 10-15 hrs at 30C with vigorous shaking.
3. Add 100 ml of prewarmed LB10:5:5 media to the culture and 1 hr more culture.
4. 4C – Collect cells by centrifugation (5000 x g for 10 min).
5. 4C – Discard medium, resuspend cells in 5 ml 15% glycerol, transfer cells to five or more 1.5 ml E-tubes.
6. 4C – Wash the cells 4 times by 5000 x g for 2 min; each time add 1 ml of 15% glycerol.
7. Measure the volume of cell pellets and resuspend the pellets in 10-volume of 15% glycerol.
8. Should have at least 5 ml of resuspended cells and aliquot and store at -70C for a few years.