Team:TU Darmstadt/Protocols/Chemically competent cells
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* ice cold 100mM CaCl<sub>2</sub> | * ice cold 100mM CaCl<sub>2</sub> | ||
=== Procedure === | === Procedure === | ||
- | # | + | # Inoculate 2 mL of LB-Media with an E. coli colony and incubate at 37 °C overnight |
- | + | # Inoculate 200 mL LB with the preculture | |
- | # | + | # Incubate at 37°C and 150 rpm until an OD<sub>600</sub> of 0.4-0.6 is reached |
- | # | + | # Incubate cells on ice for 15 min |
- | # | + | # Centrifuge the culture at 4°C and 3000 x g for 10 min (the following steps are carried out on ice) |
- | # | + | # Resuspend cell pellet in 10mL ice cold 100 mM CaCl<sub>2</sub> (Do not vortex!) |
- | # | + | # Incubate on ice for 1 hour |
- | # | + | # Centrifuge the culture at 4°C and 3000 x g for 10 min |
- | # | + | # Resuspend cell pellet in 10mL ice cold 100 mM CaCl<sub>2</sub> |
- | # | + | # Incubate on ice for 1 hour |
- | # | + | # Centrifuge the culture at 4°C and 3000 x g for 5 min |
- | # | + | # Resuspend cell pellet in 2mL ice cold 100 mM CaCl<sub>2</sub> and 15 % (v/v) glycerine |
- | # | + | # Incubate on ice for 30 min |
- | # | + | # Aliquot the cells à 100µL |
- | # | + | # Store at -80°C |
- | # | + | |
+ | === 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 | ||
+ | |||
== References == | == References == | ||
* Mandel, M. and Higa, A.: Calcium-dependent bacteriophage DNA infection. J Mol Biol, 1970, 53, 159-162 | * Mandel, M. and Higa, A.: Calcium-dependent bacteriophage DNA infection. J Mol Biol, 1970, 53, 159-162 |
Revision as of 19:04, 16 September 2012
Contents |
Chemically competent cells
The transformation of E. coli with plasmid DNA via heatshock transformation requires chemically competent cells.
Materials
Equipment
- -80°C freezer
- Incubation shaker
- Centrifuge (cooling cababilities required!)
- photometer
- Ice water bath
Chemicals & consumables
- Ice and/or liquid nitrogen
- Falcon tubes
- dYT Medium (50 ml p.c.)
- ice cold 100mM CaCl2
Procedure
- Inoculate 2 mL of LB-Media with an E. coli colony and incubate at 37 °C overnight
- Inoculate 200 mL LB with the preculture
- Incubate at 37°C and 150 rpm until an OD600 of 0.4-0.6 is reached
- Incubate cells on ice for 15 min
- Centrifuge the culture at 4°C and 3000 x g for 10 min (the following steps are carried out on ice)
- Resuspend cell pellet in 10mL ice cold 100 mM CaCl2 (Do not vortex!)
- Incubate on ice for 1 hour
- Centrifuge the culture at 4°C and 3000 x g for 10 min
- Resuspend cell pellet in 10mL ice cold 100 mM CaCl2
- Incubate on ice for 1 hour
- Centrifuge the culture at 4°C and 3000 x g for 5 min
- Resuspend cell pellet in 2mL ice cold 100 mM CaCl2 and 15 % (v/v) glycerine
- Incubate on ice for 30 min
- Aliquot the cells à 100µL
- Store at -80°C
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
References
- Mandel, M. and Higa, A.: Calcium-dependent bacteriophage DNA infection. J Mol Biol, 1970, 53, 159-162