Bacterial Transformation - Electroporation

Jeff Lawrence

Preparation of Electrocompetent cells

1. Dilute a 5 mL cell culture into 1 L LB and grow to late log phase. Chill cells on ice.

2. Centrifuge cells in GSA rotor @4000 krpm for 15 min. Discard supernatant.

3. Resuspend cells in 500 mL ice cold ddH₂O.

4. Centrifuge cells in GSA rotor @4000 krpm for 15 min. Discard supernatant.

5. Resuspend cells in 250 mL ice cold ddH₂O.

6. Centrifuge cells in GSA rotor @4000 krpm for 15 min. Discard supernatant.

7. Resuspend cells in 30 mL ice cold ddH₂O.

8. Centrifuge cells in SS34 rotor @4000 krpm for 8 min. Discard supernatant.

9. Resuspend cells in 2 mL ice cold ddH₂O.

Electroporation

1. Set Pulse Generator to 2.5 kV, 21 µF

2. Add 1 mL DNA in ddH₂O to 40 µL Electrocompetent cells.

3. Place cells in electroporation cuvette (2 mm gap) and immediately electroporate. Decay time should be around 4.6 msec.

4. Immediately add 500 µL ice cold SOC. SOC is 20 mM glucose in SOB:

	2.0 %	Tryptone	20.0 g	BactoTryptone
	0.5 %	Yeast Extract	5.0 g	Yeast Extract
	10.0 mM	NaCl		2 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
				970 mL	ddH₂O

5. Mix gently and incubate on ice for 5 min.

6. Transfer to a culture tube and incubate at 37° for 1 hr with shaking.

7. Plate 50 µL cells and grow overnight.

Last Update: Thursday June 19 2014

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Eric Kofoid eckofoid at ucdavis.edu