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CloningCloning Methods

Preparation of chemically competent E.coli cells

Materials

TB buffer

  • 10 mM HEPES pH 6.7
  • 15 mM CaCl2
  • 55 mM MnCl2
  • 250 mM KCl

Mix all component except MnCl2 and adjust the pH to 6.7 with KOH. Then add the MnCl2 and filter sterilize the mixture using a 0.22-µm filter.

LB medium:

  • 20 g/L bacto-tryptone
  • 5 g/L yeast extract
  • 5 g/L NaCl

SOB++ medium

  • 20 g/L bacto-tryptone
  • 5 g/L yeast extract
  • 0.5 g/L NaCl
  • 0.186 g/L KCl
  • 10 mM MgCl2
  • 10 mM MgSO4

Heat sterilize the medium and add sterile MgCl2 and MgSO4 just before use.

Stock solutions:

  • 1 M MgCl2 (sterilized)
  • 1 M MgSO4 (sterilized)
  • DMSO

Procedure

1. Inoculate 3 ml LB medium with the appropriate E. coli strain and incubate the culture overnight at 37°C.
2. Add the overnight culture to 500 ml SOB++ medium and incubate the culture at 25-30°C until the absorbance at 600 nm was approx. 0.5 (between 0.4 and 0.6).

In the original paper the culture was incubated at 18°C but in our hands this did not make a difference. Good competent cells were also obtained when LB or SOC medium was used.
3. Chill the culture for at least 10 min on ice.

In the following steps, the cell suspension should be kept on ice as much as possible.
4. Centrifuge the cell suspension for 10 min at 4,500 rpm (Sorvall RCB4 rotor) or 6,000 rpm (Sorvall GSA rotor) at 4°C.
5. Gently resuspend the pellet in 100 ml ice-cold TB buffer.
6. Incubate the cell suspension on ice for 10 min.
7. Centrifuge for 10 min at rpm (Sorvall SS-34 rotor) at 4°C.
8. Gently resuspend the pellet in 18.6 ml ice-cold TB buffer and add 1.4 ml DMSO.
9. Incubate the cell suspension on wet ice for at least 10 min.
10. Aliquot the cell suspension at 600 µl per tube.
11. Shock-freeze the cell suspension in liquid nitrogen and store the tubes at -80° or in liquid nitrogen.

At -80°C the cells will be competent for at least 6 months. In liquid nitrogen they will stay competent indefinitely.

Remarks:

  • The transformation efficiency of the competent cells can be determined using 5 ng of pure plasmid DNA
    and 200 µl competent cell suspension (see transformation protocol).
  • This will give approx. 1 x 108 transformants per µg plasmid DNA for DH5a.

Reference

Inoue et al. (1990) Gene 96, 23-28.