Bacterial Transformation : RuCl Method

Jeff Lawrence

1. Inoculate 100 mL LB with 0.5 mL overnight culture in a 500 mL flask. Grow with vigorous shaking at 37° until cell density reaches 5x107 cells/mL.

2. Centrifuge 2 mL aliquots (10⁸ cells) in sterile 15 mL tubes for 10 min at 4000 g at 4°.

3. Discard supernatant. Gently resuspend cells in 1 mL of:

	10 mM 	MOPS, pH 7.0	1 mL	1 M MOPS, pH 7.0
	10 mM 	RuCl		1 mL	1 M RuCl
				98 mL	ddH₂O

4. Centrifuge for 10 min at 4000 g at 4°. Discard supernatant.

5. Resuspend cells in 1 mL of RuCl Solution:

	100 mM	MOPS, pH 6.5	10 mL	1 M MOPS, pH 6.5
	50 mM	CaCl2		5 mL	1 M CaCl2
	10 mM	RuCl		1 mL	1 M RuCl
				84 mL	ddH₂O

6. Incubate on ice for 15 min. Centrifuge for 10 min at 4000 g at 4°.

7. Discard supernatant. Gently resuspend cells in 200 µL RuCl Solution.

8. Add 3 µL fresh DMSO and up to 200 ng DNA in less than 10 µL TE.

9. Incubate on ice for 30 min.

10. Transfer to a water bath at 43° for 30 sec.

11. Add 5 mL LB and incubate at 37° for 60 min without shaking.

12. Plate on selective media.

Last Update: Thursday June 19 2014

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Eric Kofoid eckofoid at ucdavis.edu