Team:TU Darmstadt/Protocols/Metabolism
From 2012.igem.org
Protocols Metabolism
The following page give an overview of the standard protocols used by the team Metabolism in iGEM PET.erminators project 2012.
Contents |
Protocols Metabolism
In vivo
Production of chemically competent cells
Production of chemically competent cells
- Inoculate 10 ml of LB-Media with an E. coli colony and incubate at 37 °C overnight
- Inoculate 200 ml of LB-Media with 2.5 ml of the overnight culture
- Incubate the culture at 37 °C until an OD600 of 0.5
- Cool the cells on ice for 5 min
- Centrifuge the cells for 10 min at 5000 rpm and 4 °C
- Resuspend the cell-pellet in 32 ml pre-cooled CaCl2 solution (Do not vortex!)
- Add pre-cooled CaCl2 solution to 200 ml
- Let the cells repose on ice for 1 hour
- Centrifuge the cells for 10 min at 5000 rpm and 4 °C
- Resuspend the pellet in 10 ml cryo-solution
- Decant 200 µl of competent cells in a 1.5 ml tube
- Store the tube in an -80 °C freezer
Solutions
- CaCl2
- 5.55 g CaCl2
- Add di H2O to 1 L
- Sterilize by autoclaving
- Cryo solution
- 0.278 g CaCl2
- 10 ml glycerin
- Add di H2O to 50 ml
- Sterilize by autoclave