WangLab:Competent Cell Preparation

500 ml 1x TB

 * 1) combine in H2O:
 * 2) * 10mM PIPES (FW 342.4), 1.71g
 * 3) * 15mM CaCl2.2H2O (FW 147.02), 1.1g
 * 4) * 250mM KCl (FW 74.56), 9.32g
 * 5) adj PH to 6.7 with KOH, adj volume to 495 ml with water
 * 6) add MnCl2.4H2O (55mM, FW 197.9, 5.44g).
 * 7) Filter through the entire solution. Store at 4oC for up to 6 months

500ml SOC in 2L flask

 * 1) Desolve 10g Tryptone (2%)
 * 2) *	 2.5g Yeast extract (0.5%)
 * 3) add
 * 4) *       1ml  5M NaCl (filtered sterile) (final 10mM)
 * 5) *       5ml  250mM KCl (filtered sterile) (final 2.5mM)
 * 6) *	 2.5ml 2M MgSO4 (filtered sterile) (final 10 mM)
 * 7) adjust to 500 ml and autoclave
 * 8) after cool down,
 * 9) * add 2.5ml 2M MgCl2 (filtered sterile) (final 10 mM)
 * 10) * add 5ml 2M glucose (filtered sterile) (final 20 mM)

Procedure

 * day 1: streak cells on a LB plate without ampicillin
 * day 2: pick one colony and culture in 5 ml SOC 37oC.
 * day 3:
 * a. transfer 5 ml culture into 500 ml SOC, big flask
 * incubate at RT, shake at 220 RPM, (9:00am-2:30pm)
 * frequently check reading with 600 nm for cell density.
 * b. put in ice, wait for 10 min, transfer to centrifuge tubes
 * c. 4500 rpm, 10 min, spin,
 * d. pellet should be small and thin, if large, do not use it.
 * e. add 9.5ml TB, resuspend by gentle swirl, incubate 10 min in ice afterwards
 * f. spin 3000 rpm, 10 min, 4oC.
 * g. 20 ml TB for 250 ml culture with 0.6 OD, so 5-7ml for 500 ml with OD about 0.116. add 5 ml TB, gentle swirl and add 7% DMSO (350 ml), drop by drop while swirl.
 * h. wait 10 min in ice.
 * i. aliquote and put in liquid N2 immediately and keep in -80oC.