Molecular Techniques and Methods

Preparation of E.coli Competent Cells by CaCl2


Copy Right © 2001/ Institute of Molecular Development LLC



INTRODUCTION




MATERIALS AND SOLUTIONS

LB containg 10 mM MgCl2

Ice cold 75 mM CaCl2

50% Glycerol (sterile)



PROCEDURES



1. Start 2 ml overnight culture from a single colony.

2. Next day, inoculate 1 ml of overnight culture to 80 ml LB containing 10 mM MgCl2 (sterile).

3. Grow to O.D.600 = 0.4 (2.5-3 hours).


  • All steps should be done in ice and srerile condition.

    4. Cool in ice for 5 min.

    5. Centrifuge for 10 min, 3,000 rpm at 4oC.

    6. Discard supernatant.

    7. Resuspend bacterial pellet in 34 ml of ice cold, sterile 75 mM CaCl2.

    8. Vortex to resuspend completely and incubate in ice for 40 min.

    9. Centrifuge for 10 min, 3,000 rpm at 4oC.

    10. Resuspend bacterial pellet in 3.4 ml of ice cold, sterile 75 mM CaCl2.

    11. Add 1.7 ml of sterile 50% glycerol.

    12. Immediately, dispense 50 ul each in microfuge tubes in ice and store at -70oC.
  • Important - Do not keep competent cells at room temperature.




    NOTES
  • Usually good for up to 6 months.



    KIT INFORMATION


    REFERENCES


    Please send your comment on this protocol to "editor@MolecularInfo.com".

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