Bacterial Transformation - Cohen method
Jeff Lawrence
1. Inoculate 50 mL LB with 0.5 mL of a fresh overnight culture. Grow to OD 0.6.
2. Chill cells on ice. Centrifuge at 7000 krpm for 10 min in SS34 rotor at 4°.
3. Discard supernatant. Resuspend cells in 50 mL ice cold 100 mM MgCl2.
4. Centrifuge at 7000 krpm for 10 min at 4°. Discard supernatant.
5. Resuspend cells in 25 mL ice cold 100 mM CaCl2.
6. Centrifuge at 7000 krpm for 10 min at 4°. Discard supernatant.
6. Resuspend cells in 2.5 mL ice cold 100 mM CaCl2.
7. Add 200 µL cells to 1-100 ng DNA in 100 µL of:
20 mM Tris, pH 8.0 2 mL 1 M Tris, pH 8.0
20 mM NaCl 400 mL 5 M NaCl
1 mM EDTA 200 mL 500 mM EDTA, pH 8.0
97 mL ddH2O
8. Incubate on ice for 30 min.
9. Transfer tubes to 42° for 30 sec.
10. Rechill on ice for 2 min.
11. Add 3 mL pre-warmed LB. Incubate with shaking at 37° for 60 min.
12. Plate on selective media.
Last Update: Thursday June 19 2014
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Eric Kofoid
eckofoid at ucdavis.edu