Team:Waterloo/Notebook/Protocols/Competent Cells

 Recipe for Competent Cells 

Materials  Frozen stock of DH5 alpha 250mL Erlenmeyer Flask (w/ caps) LB tubes Sterile LB

Instructions  1. From frozen stock DH5 alpha, inoculate 5mL LB tubes and grow overnight at 37˚C 2. Autoclave 250 mL Erlenmeyer flask with 50 mL LB broth for tomorrow. Don’t forget to loosen to cap if you are not using tin foil. 3. Next morning: add culture into the autoclaved 250mL Erlenmeyer Flask with 50 mL LB broth. 4. Grow at 37 ˚C for 2-3 h to OD 0.4-0.6 i.e. log phase. Ask a lab leader to look at it to save time, or otherwise use a spectrophotometer to read it at absorbance at 600nm. 5. Transfer cultures to a falcon tube and spin at 4000 rpm for 15 min at 4˚C 6. Resuspend in 20 mL of 0.1 M MgCl2 (preferred) or CaCl2 (note: swirl until cells are completely resuspended, do not vortex.) 7. Spin again. 8. Resuspend in 10mL of 0.1 M CaCl2 9. Incubate on ice for 4 hours 10. Spin again and prepare CaCl2-glycerol solution.

___ Culture tubes (___+1) x 0.86mL CaCl2 (___+1) x 0.14mL 100% glycerol

11. Do the following in 4˚C walk-in: - Resuspend a culture tube with 2mL of the CaCl2-glycerol solution, and then resuspend a second culture tube with this. - Aliquot into 200 µL or 500µL