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__NOTOC__{{UT-Tokyo_CSS3}}{{UT-Tokyo_Head}} <h1>Producing Competent Cell</h1> <h2>Preperation</h2> *・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 <h2>Procedure</h2> *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 {{UT-Tokyo_Foot}}

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