Preparation of Competent Cells

Materials
  1. LB
  2. Fresh agar plate of E. coli colonies
  3. 50ml Falcon tubes
  4. Incubator shaker 37oC
  5. -80oC freezer
  6. cryo-racks
  7. 1M MgCl2, sterile
  8. 30% Glycerol, sterile
  9. DMSO
  10. 1X DYT:
  11. 10g Tryptone
  12. 10g Yeast Extract 5g NaCl 2 pellets of NaOH
  13. Dissolve in 1L H2O. Autoclave 20 min on liquid cycle.

    14. Alternitively Terrific Broth [MPSF] can be used and the transformation effeciency of the competent cells will be the same or even better.

  14. TSS:
    • 2.8g Tryptone 2.8g Yeast Extract 1.4g NaCl 27.5g PEG 8000
    • Add H2O to 250ml. Autoclave 20 min on liquid cycle.
  15. Dry ice-Ethanol bath:
    • Scrap some dry ice to powder and small pieces into absolute ethanol in a microsample tube rack (Fisher#05-561-10).
  16. Wet ice both
Procedure:
  1. Grow a 5ml overnight culture of bacteria.
  2. Add 0.5-1ml overnight culture to 100ml DYT (or Terrific Broth) in a 250ml flask. Read OD600 with DYT (Terrific Broth) as background.
      Check OD periodically.
  3. Put the following solutions on ice:
       TSS
       1M MgCl2
       30% glycerol
  4. Prepare a dry ice-ethanol bath.
  5. When the bacteria have reached an OD of 0.5, transfer the culture to two 50ml Falcon tubes. Spin 10 min at 2000rpm at 4C.
  6. Pour off the liquid and keep the pellets on ice.
  7. Resuspend both pellets togather in 1.8ml ice cold TSS.
  8. Add 100ul 1M MgCl2.
  9. Add 100ul DMSO.
  10. Swirl solution gently. keep on ice.
  11. Add 2ml sterile 30% glycerol. Swirl gently.
  12. Aliquot competent cells at 100ul into 1.5ml eppendorf tubes. Freeze cells in dry ice-ethanol bath immediately.
  13. Transfer tubes with compotent cells into pre-cooled freezing box (label the box before pre-cooling is recommended). Store at -80C.
 
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