Heat shock competent cells

Responsible persons

If you have any troubles please contact Annett or Akira.

TB buffer

Ingredients

10 mM HEPES pH 6.7, 15 mM CaCl2, 55 mM MnCl2, 250 mM KCl
Make fresh. 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.

Procedure

1. 1. Streak out the strain on LB plate (containing appropriate antibiotic if needed) and grow overnight at 37°.
   2. Inoculate 3 ml LB medium with a single colony of the appropriate E. coli strain and incubate the culture overnight at 37°C.
2. Add 2ml overnight culture to 400 ml SOB++ medium in 2.8l flask and incubate the culture at 18°C until the absorbance at 600 nm is approximately 0.6 (between 0.4 and 0.8). For DH5alpha it takes around 24hr-30hr.
3. Chill the culture for at least 10 min on ice.
4. Centrifuge the cell suspension (33ml each in 50ml tubes x12) for 10 min at 2500g (JA12 rotor) at 4°C. Invert the tube on a tissue and drain to remove all media.
5. Gently resuspend the pellet in 180 ml (15ml / tube) ice-cold TB buffer.
6. Centrifuge the cell suspension (combine two tubes into one, 30ml/tube) for 10 min at 2500g (JA12 rotor) at 4°C. Invert the tube on a tissue and drain to remove all media.
7. Gently resuspend the pellet in 90 ml (15ml / tube) ice-cold TB buffer.
8. Centrifuge the cell suspension (combine two tubes into one, 30ml / tube) for 10 min at 2500g (JA12 rotor) at 4°C. Invert the tube on a tissue and drain to remove all media.
9. Gently resuspend the pellet in 30 ml (10ml / tube) ice-cold TB buffer.
10. Add 2.35ml (0.75ml / tube) DMSO and mix gently.
11. Incubate the cell suspension on wet ice for at least 10 min.
12. Aliquot the cell suspension (25 µl per tube).
13. Shock-freeze the cell suspension in liquid nitrogen and store the tubes at -80° or in liquid nitrogen.

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NOTE:

• At -80°C the cells will be competent for at least 6months. In liquid nitrogen they will stay competent indefinitely.
• This will give approx. 1 x 108 transformants per µg plasmid DNA for DH5a.

References

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