Preparation of ultra-competent E. coli cells for transformation

Based on Inoue et al (1990), Gene, 96:23-28, with modifications.
  1. Culture cells (DH5a in my case) on LB agar plate at 37oC overnight.
  2. Pick up 10 -12 large colonies and culture in 250ml SOB in a 1L flask, 19oC with vigorous shaking to OD600=0.5 (normally it takes 24-36hrs)
  3. Place the flask in ice for 10 min.
  4. Pelleting the cell by spining at 4000rpm for 10 min at 4oC.
  5. Gently resuspend the cell in 80ml ice-cold TB and store on ice for 10 min.
  6. Spin at 4000rpm for 10 min at 4oC
  7. Gently resuspend the pellet in 20ml ice-cold TB and 1.4ml DMSO (the DMSO needs to be stored at -20oC o/n before use).
  8. Aliquote the cell to 50 to 500ul for transformation or store at -70oC