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Team:Waterloo/Notebook/Protocols/Competent Cells
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Recipe for Competent Cells
Materials
Frozen stock DH5a
250mL Erlenmeyer Flask (w/ caps)
LB tubes
Sterile LB
Instructions
1. From frozen stock DH5a, 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
