Preparation of E. coli competent cell

Preparation of E. coli competent cell

Material

SOB medium

Reagent Vol
Bacto tryptone 20 g
Yeast extract 5 g
5 M NaCl 2 ml
2 M KCl 1.25 ml
H2O to 990 ml
  1. Autoclave medium shown in table.
  2. Add 10 ml autoclaved 2 M Mg2+ sol (1 M MgSO4·2H2O/250 mM KCl).
  3. Store at 4C.

Day 1

  1. Seed competent cell on LB(-) plate.
  2. Incubate at 37C for o/n.

Day 2

  1. Pick up E. coli colony by yellow tip.
  2. Put tip into 5 ml LB(-) medium in 15 ml tube.
  3. Culture at 250 rpm at 37C for o/n.

Day 3

  1. Add 1 ml precultured medium to two 125 ml (total 250 ml) SOB medium.
  2. Culture at 250 rpm at 18C for o/n.

Day 4-5

OD600 become 0.4-0.8 (0.6 is best) after 24-48 h of culture.

  1. Incubate flask on ice for 10 min.
  2. Move medium to 50 ml tube.
  3. Centrifuge at 4500 rpm at 4C for 10 min.
  4. Remove supernate.
  5. Collect cells with washing by two 42 ml (total 84 ml, 1/3 vol of SOB medium) transformation buf.
  6. Centrifuge at 4500 rpm at 4C for 10 min.
  7. Remove supernate.
  8. Suspend all cells in 10 ml transformation buf.
  9. Add 750 ul DMSO.
  10. Incubate on ice for 10 min.
  11. Aliquote in 1.5 ml tube (100-200 ul/tube).
  12. Freeze in liquid N2.
  13. Store at -80C.