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, 19-Jun-2014 11:50:36 PDT
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Eric Kofoid eckofoid at ucdavis.edu