Team:INSA-Lyon/Protocols/Competent cells

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Revision as of 13:20, 10 September 2010







Protocols


Currently under construction



  1. Competent cells
  2. Transformation
  3. DNA extraction
  4. Digestion
  5. Ligation
  6. Measure of temperature and shaking speed influence
  7. Measure of osmotic pressure influence
  8. Granules extraction and intein cleavage
  9. Biofilms quantification
  10. 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 deg

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