Transformation (standard protocol for DH5a and BL21)

Work under sterile conditions.
  1. Thaw the cells until just liquid ca. 3 min. Put on ice for 5-10 min. Transfer cells into round-bottom polypropylene culture tubes. Use 50 µl DH5a per simple plasmid transformation, 50-100 µl DH5a for ligations. Use 100-200 µl BL21 for plasmid transformations.
  2. Add the DNA-solution in a volume equal to or less than 1/4 volume of cells and at a concentration of < 0.1 µg per 200 µl of cells.
  3. Leave on ice for 20 min.
  4. Incubate at 42°C for 60 sec.
  5. Stand on ice for 1-2 min.
  6. Add a minimum of 4 volumes of LB at room temperature and shake gently (180 rpm) at 37°C for 50-60 min, not longer!
  7. Plate on LB-Agar with respective antibiotic (50 µg/ml ampicillin or 25-50 µg/ml kanamycin.

For Plasmid transformations, plate 100 µl neat, 100 µl of a 1:10 dilution in LB, and 100 µl of a 1:100 dilution.

For ligations: If 50 µl cells were transformed, plate all. If 200 µl cells were transformed, plate 200 µl neat and 200 µl of a 1:10 dilution and the remaining cells on separate plates.