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IN VIVO SYSTEMS > BUGS > Competent Cells

> Preparation of Competent E coli

> DMSO Method

> One Step Preparation

Preparation of Competent E. Coli

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This protocol is a larger scale variant of one due to Mike Scott, Department of Neurology, UCSF.

  1. Streak out the desired strain on an LB plate. Pick colony and grow in 3mL LB over night. (If you are using SureTM cells, then use LB+Tetracycline.)
  2. The next day, inoculate 100 ul into 20 ml TYM broth in a 250 ml flask. Preheat all broth solutions to 37ºC if you want to be done before midnight.
  3. Grow the cells to midlog phase (OD600 between 0.2 - 0.8), pour into 2 L flask containing 100 ml TYM, continue vigorous agitation until cells grow to 0.5 - 0.9 OD, then dilute again to 500 ml in the same vessel.
  4. When cells grow to an OD600 of 0.6 put the flask in ice-water, and swirl contents gently to assure rapid cooling. When culture is cool, pellet the cells  (spin 2.5 krpm for 15 minutes in the JA-10).
  5. Pour off the supernatant, resuspend the pellet in 100 ml cold TfB I  by gentle shaking on ice. Re-pellet the cells in the same bottle (spin 2.5 krpm for 8 minutes in the JA-10 rotor).
  6. Pour off supernatant, and resuspend the pellet in 20 ml cold TfB II by gentle shaking on ice.
  7. Dispense in 250 ul aliquots in prechilled microfuge tubes, freeze in liquid nitrogen, and store at -70oC. For transformation protocols, click here

MEDIA + BUFFERS

(1L) TYM Broth
2% bactotryptone 20 g
0.5% yeast extract 5 g
0.1M NaCl 5.84 g
10mM MgSO4 2.46 g
Water to 1L

Separate into 20 ml, 100 ml, 400 ml volumes and autoclave.

(250mL) TfBI
30mM KoAc 0.736g
50mM MnCl2 2.47g
100mM KCl 1.86g
10mM CaCl2 dihydrate 0.367g
15% (v/v) glycerol 37.5mL
 
(100mL) TfBII
10mM Na-MOPS pH 7.0 (stock: 50mM pH 7.0) 20 mL
75mM CaCl2 dihydrate 1.1 g
10mM KCl 0.0745 g
15% glycerol 15 mL

TfBI and II : DO NOT AUTOCLAVE . FILTER WITH A 0.2 um FILTER AND STORE  at 4o C.