Team:UT-Tokyo/Producing Competent Cell

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UT-Tokyo

Producing Competent Cell

Preperation

  • ・SOB medium (2.0% tryptone, 0.5% yeast extract, 10 ml NaCl, 2.5 ml KCl. Add one hundredth of medium of 1M Mg solution (500 mM MgCl_2, 500 mM MgSO_4) before using)
  • ・50 ml TB (10 mM PIPES-KOH (pH 6.7), 15mM CaCl_2, 250 mM KCl, 55 mM MnCl_2)
  • ・DH5\alpha strain, LB medium

Procedure

  • 1. Incubate O/N at 37 degrees C in LB medium
  • 2. Add 2-5 ml O/N culture to 200 ml SOB medium
  • 3. Incubate at 18 degrees C until OD600 turns to 0.4
  • 4. Put culture on ice for 10 minutes
  • 5. Centrifuge (4000 g, 5 minutes, 4 degrees C) with four 50 ml tube
  • 6. Remove supernatant
  • 7. Resuspend slowly with 15 ml TB
  • 8. Put culture on ice for 15 minutes
  • 9. Repeat procedure 5 & 6
  • 10. Resuspend slowly with 7 ml TB
  • 11. Put culture on ice for 5 minutes
  • 12. Add 490 ul DMSO, mix well and put it on ice for 15 minutes
  • 13. Repeat procedure 12
  • 14. Divide cells into 100 ul aliquots and store at -80 degrees C after freezing by liquid nitrogen