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Team:TU Darmstadt/Protocols/Metabolism
From 2012.igem.org
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| - | <span style="font-size:200%;"><span style="color:#00689D;">Protocols</span></span> | + | <span style="font-size:200%;"><span style="color:#00689D;">Protocols Metabolism</span></span> |
The following page(s) give an overview of the standard protocols used by the team Metabolism in iGEM PET.erminators project 2012. | The following page(s) give an overview of the standard protocols used by the team Metabolism in iGEM PET.erminators project 2012. | ||
| + | |||
| + | ==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 CaCl<sub>2</sub> solution (Do not vortex!) | ||
| + | * Add pre-cooled CaCl<sub>2</sub> 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''' | ||
| + | * CaCl<sub>2</sub> | ||
| + | **5.55 g CaCl<sub>2</sub> | ||
| + | **Add di H<sub>2>/sub>O to 1 L | ||
| + | **Sterilize by autoclaving | ||
| + | |||
| + | *Cryo solution | ||
| + | ** 0.278 g CaCl<sub>2</sub> | ||
| + | ** 10 ml glycerin | ||
| + | ** Add di H<sub>2>/sub>O to 50 ml | ||
| + | ** Sterilize by autoclave | ||
Revision as of 14:10, 9 September 2012
Protocols Metabolism
The following page(s) give an overview of the standard protocols used by the team Metabolism in iGEM PET.erminators project 2012.
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 H2>/sub>O to 1 L
- Sterilize by autoclaving
- Cryo solution
- 0.278 g CaCl2
- 10 ml glycerin
- Add di H2>/sub>O to 50 ml
- Sterilize by autoclave
