Team:Tuebingen/Notebook/Protocols/chemo-competentcells

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Procedure

General advice:
It is advantageous to scale up the volume in order to yield more aliquots! Use additional flasks when necessary.

1. Inoculate 25 mL SOB-medium with one single colony from a fresh plate.
2. Incubate for 8 h at 37 °C and 250 rpm.
3. Inoculate three falcon tubes containing 100 mL SOB-medium each with 1 mL, 2 mL and 4 mL of the prepared pre-culture (i.e. 100 mL SOB + 1 mL pre-culture; 100 mL SOB + 2 mL pre-culture, and 100 mL SOB + 4 mL pre-culture).
4. Incubate over night at 18 to 22 °C and 200 rpm.
5. On the next day, check OD600. At OD600 = 0.55, put culture on ice for 10 min.
6. Centrifuge cells at 2500 g and 4 °C for 10 min. Discard supernatant completely.
7. Resuspend cell pellet in 30 mL Inoue buffer at 0 °C.
8. Centrifuge cells at 2500 g and 4 °C for 10 min. Discard supernatant completely.
9. Repeat the previous two steps.
10. Resuspend cells in 8 mL Inoue buffer at 0 °C. Add 1.5 mL DMSO and incubate for 10 min.
11. Aliquot cells à 100 µL in Eppendorf-tubes and freeze in liquid nitrogen. Store at -80 °C.