Quint Lab:electrocompetent cells e.coli

From OpenWetWare

Revision as of 10:59, 28 April 2008 by Marcel Quint (Talk | contribs)
(diff) ←Older revision | Current revision (diff) | Newer revision→ (diff)
Jump to: navigation, search

home        research        people        papers        teaching        join us        internal        pictures        contact       


Electro competent Escherichia coli cells

this protocol works fine with E.coli DB3.1 cells

Materials:

  • 10 mL LB-Medium (1% Bacto Trypton; 0,5 % Yeast Extract; 0,5% NaCl)
  • 1L LB-light Medium (1% Bacto Trypton; 0,5 % Yeast Extract; 0,25% NaCl)
  • 2L cooled bidest H2O
  • 200 mL cooled, sterile-filtered 10% Glycerol
  • box with ice-water for 2-litre-flask
  • 4 pre-cooled 250 mL (or 2x500 mL) bins for centrifugation
  • 2 pre-cooled 50 mL Falcons
  • centrifuge pre-cooled to 2°C (max. 4°C)
  1. inoculate 10 mL LB with bacterial stock; incubate over night at 37°C and 200 rpm
  2. inoculate 1 L LB-light in 2-litre-flask with 10 mL preculture
  3. incubate until OD600 0,4-0,6 (~5 h)


  • from now on everything is done at 2-4°C (best in a cold room)
  1. cool 1L-culture 10-15 minutes in ice water (shake sometimes)
  2. divide culture into 4 cooled 250 mL bins for centrifugation
  3. centrifuge 20min @ 2°C, 4200 rpm
  4. discard supernatant
  5. resuspend pellet in 5 mL bidest H2O
  6. add bidest H2O up to 250 mL
  7. centrifuge 20min @ 2°C, 4200 rpm
  8. discard supernatant immediately
  9. resuspend pellet in residual supernatant
  10. add bidest H2O up to 250 mL
  11. centrifuge 20min @ 2°C, 4200 rpm
  12. discard supernatant immediately
  13. resuspend pellet in residual supernatant
  14. transfer suspension in 50 mL Falcons
  15. add 10% Glycerol up to 50 mL
  16. centrifuge 10 min @ 2°C, 4000 rpm
  17. discard supernatant
  18. estimate volume of the pellet; fill up with equal volume of 10% Glycerol
  19. resuspend pellet on ice; ‘’’don´t vortex!!’’’ (just shake cautiously)
  20. divide cells into 200 or 400 µL Allicots (use 1,5 mL Eppis)
  21. freeze in liquid N2 or dry-ice
  22. store @ -80°C

Transformation via electroporation

  1. add 0,5-2 µL plasmid to 50 µl electrocompetent cells
  2. electroporate at U=2,5 kV, C= 25 µF, R = 200 Ώ
  3. transfer transformation reaction to 450 µL SOC-Medium and shake 1 h at 37°C
  4. centrifuge 2 min @ 800 rpm and plate on selective LB-Medium
Personal tools