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From 2010.igem.org

Competent cells (From Morten Nørholm at the Department of Biochemistry & Biophysics Stockholm University)

1. Add 5 ml LB in two 50 ml falcon tubes. Add the top of a tip bacteria into the two 5 ml LB. Grow ON in shake incubator 37 degree C.

2. Subculture each 5 ml of starterculture into two 400 ml pre-warmed LB. Grow at 37 degree C until OD reaches 0.6.

3. Put cells on ice for 20 min.

4. Harvest cells at 4000 rpm for 20 min, 4 degree C.

5. Discard supernatant if it looks clear (or spinn longer if it is cloudy).

6. Resuspend pellet carefully in 500 50 mM CaCl2 for a 1000 ml cell culture (1/2 the orifinal volume).

7. Put cells on ice 20 min.

8. Repeat step 5.

9. Resuspend cells in 16 ml CaCl2 + 15% Glycerol for a 800 ml starter culture (1:50 volume)

10. Put metal blocks in -80 degree C.

11. Snapfreeze 100 mikroL aliquots in ice-cold Epps (in pre-chilled blocks). Store in -80 degree C freezer.