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