Team:Mississippi State/Making Competent Cells

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Home The Team The Project Parts Submitted to the Registry Modeling Notebook
  • To make competent cells(XL1-Blue), do exactly as follows:
  1. Add 2ml LB Broth to Culture Tube.
  2. Add 1ul antibiotic for every 1ml LB(in this case, 2ul of Tetracycline).
  3. Get cell stock from -80C freezer.
  4. Scrape cells from top with tip of pipette.
  5. Add to LB Broth.
  6. Label (Culture name, date, iGEM)
  7. Put in 37C shaker overnight.
  8. Remove next morning.
  9. Get pipette gun from 1st drawer from left under lab bench closest to door.
  10. Get 10ml tube from lab bench opposite 1st lab bench.
  11. Add 25ml LB Broth to autoclaved flask(KEEP THE FOIL COVER!).
  12. Add 25ul Tet (in IGEM 2007 Box, -20C freezer)
  13. Add 250ul cells from overnight culture.
  14. Cover with aluminum
  15. Put in 37C shaker for 2hr.
  16. Check cell density
  17. Pour into centrifuge tubes.
  18. Centrifuge 10-15min. in SS34 model, which is setting 05 for 15min at 5000rpm and +4C.
  19. Pour out supernatant into same flask, autoclave this flask before disposal.
  20. Resuspend cells in CaCl, using half previous volume: for example, if you originally had cells in 10ml LB, you would use 5ml CaCl.
  21. Centrifuge again at same settings.
  22. Pour out supernatant.
  23. Resuspend in CaCl, this time using 2ml of CaCl.
  24. Store on Ice for 1hr.
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