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Team:INSA-Lyon/Protocols/Competent cells
From 2010.igem.org
Protocols
Choose a protocol to read its description :
- Competent cells
- Transformation
- DNA extraction
- Digestion
- Ligation
- Measure of temperature and shaking speed influence
- Measure of osmotic pressure influence
- Granules extraction and intein cleavage
- Biofilms quantification
- Extra
Competent cells
- Inoculate about 5 mL sterile LB broth with the appropriate chassis
- Grow the cells on a shaker at 37°C overnight
- In the morning, inoculate about 100 mL sterile LB broth with the 5 mL-preculture (1:200).
- Grow the cells on a shaker at 37°C until they reach an OD at 600 nm of 0.4 (about 108 bacteria/mL)
It is possible to inoculate directly in 100 mL until they reach an OD at 0,4.
- Transfer the culture in 40 mL falcon tube. Chill the cells on ice for 10 min.
From now on always work on ice - Spin the cells down at 5000 rpm for 10 min at 4°C
- Remove the supernatant using a sterile pipet
- Resuspend each tube with 1/2 volume of sterile and cold CaCl2 100 mM (25 mL).
Keep on ice for 30 min
Spin at 5000 rpm for 5 min at 4°C - Resuspend each tube gently (by pipeting) with about 1/10 volume of CaCl2 100 mM, sterile cold glycerol 15% (2,5 mL CaCl2 100 mM + 1,5 glycerol 40%)
- Aliquot 0.2 ml (200 µl) of the solution using a sterile pipet into 1.5 ml eppendorf tubes which have been chilled on ice
Use in the day or store the cells at -80 °C
