Bacterial Transformation - Hanahan Method

Jeff Lawrence

1. Inoculate 1 mL SOB with a single colony. Vortex and use to innoculate 50 mL SOB. Grow to Klett 140, or OD 0.5 @ 550 nm (5x108 cells/mL). SOB is :

	2.0 %	Typtone		20 g	Bactotyptone
	0.5 %	Yeast Extract	5 g	Yeast Extract
	10.0 mM	NaCl		2.0 mL	5 M NaCl
	2.5 mM	KCl		2.5 mL	1 M KCl
	10.0 mM	MgCl2		10.0 mL	1 M MgCl2
	10.0 mM	MgSO4		10.0 mL	1 M MgSO4
				930 mL	ddH₂O

2. Chill on ice 15 min. Centrifuge at 4000 g for 12 min at 4°. Resuspend in 16 mL cold FSB:

	10 mM	KOAc		10 mL	1 M KOAc, pH 7.0
	100 mM	KCl		100 mL	1 M KCl
	45 mM	MnCl2		45 mL	1 M MnCl2
	10 mM	CaCl2		10 mL	1 M CaCl2
	3 mM	HACoCl2		30 mL	100 mM HACoCl2
	10 %	Glycerol	200 mL	50% Glycerol
				605 mL	ddH₂O

3. Chill on ice 15 min. Centrifuge at 4000 g for 12 min at 4°. Resuspend in 4 mL cold FSB.

4. Add 140 µL fresh DMSO. Chill on ice for 5 min.

5. Add 150 µL of DTT Solution:

	2.25 M	DTT		900 µL	2.5 M DTT
	40.0 mM	KOAc, pH 6.0	40 µL 	1 M KOAc, pH 6.0
				60 µL	ddH₂O

6. Chill on ice 10 min. Add 140 µL fresh DMSO and incubate on ice 5 min.

7. Aliquot 200 µL cells into chilled 5 mL tubes. Add up to 100 ng DNA in less than 10 µL TE. Swirl and incubate on ice for 30 min.

8. Place samples at 42° for 90 sec. Rechill on ice for 2 min.

9. Add 800 µL SOC:

	1 X	SOB	100 mL	SOB
	20 mM	Glucose	200 mL 	1 M Glucose

10. Incubate at 37° for 60 min with swirling. Plate on selective media.

Last Update: Thursday June 19 2014

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Eric Kofoid eckofoid at ucdavis.edu