Molecular Techniques and Methods

Preparation of E.coli Competent Cells by RbCl Method


Copy Right © 2001/ Institute of Molecular Development LLC



INTRODUCTION




MATERIALS AND SOLUTIONS

TFB1 Buffer (100 ml)
100 mM RbCl
50 mM MnCl2
30 mM KAc
10 mM CaCl2
15% Glycerol

  • Adjust pH to 5.8.
  • Sterile-filtered by 0.2 um membrane.


    TFB2 Buffer (50 ml)
    10 mM MOPS
    10 mM RbCl
    75 mM CaCl2
    15% Glycerol
  • Adjust pH to 8.0.
  • Autoclave.




    PROCEDURES

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

    2. Inoculate 1 ml of overnight culture to 100 ml LB.

    3. Grow to A600 = 0.5 (usually 2-3 hours).

    4. Centrifuge for 5 min., 4,500 rpm at 4oC.

    5. Discard supernatant.

    6. Resuspend the cells in cold TFB1 Buffer (30 ml/ 100 ml culture).

    7. Keep on ice for 90 min.

    8. Centrifuge for 5 min., 4,500 rpm at 4oC.

    9. Resuspend the cells in ice cold TFB2 Buffer (4 ml/ 100 ml culture).

    10. Dispense 100 ul each, snap freeze in liquid nitrogen and store at -70oC.




    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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