Transformation of electrocompetent cells by electroporation

Dilute 2 ul of ligation mix with 2 ul of water. Mix 1 ul of diluted ligation reaction with 20 ul of electro-competent cells. After electroporation transfer cells into the 500 ul of SOC media. Incubate 1 hour at 37 °C with gentle shaking. Plate 10 and 100 ul of cells on LB plates with ampicillin (100mkg/ml), X-gal and IPTG. Incubate overnight at 37 °C.

LB/Am/X-gal/IPTG plates:

2.5 g Tryptone

5 g Sodium chloride

2.5 g Yeast

7.5 g Agar

Add deionized water to 500 ml final volume. Autoclave.

Add 500 ul of ampicillin (100mg/ml in water), 50 ul of IPTG (200mg/ml in water) and 400 ul of X-gal (50mg/ml in N,N’-dimethylformamide).

Preparation of electro-competent E.coli cells.

Electromax DH10B E.coli strain could be used to prepare electro-competent cells.

Plate cells on fresh LB plate without antibiotic to get a single colony. Use a fresh colony of cells to inoculate 50 ml of SOB media (w/o magnesium). Grow overnight culture with vigorous aeration at 37C.

Transfer 0.25 ml of overnight culture into 250 ml of SOB (w/o magnesium) in 1 L flask. Grow 4-5 hours with vigorous aeration at 37C until OD (550) = 0.8.

Harvest cells by spinning at 2600 x g for 10 min. Pour off the supernatant.

Wash the cell pellet by resuspending in 250 ml of sterile ice–cold 10% glycerol (v/v). Harvest cells by spinning at 2600 x g for 15 min and carefully pour off the supernatant as soon as the rotor stops.

Repeat washing in sterile ice-cold 10% glycerol once more. Carefully pour off the supernatant as soon as the rotor stops.

Resuspend the cell pellet in 10% glycerol that remains in the centrifuge bottle (approximately1.2-1.5 ml). Usually no additional 10% glycerol needed. Cells can be used immediately or can be frozen in 100 mkl aliquots in sterile microcentrifuge tubes using a dry ice-ethanol bath. Store frozen cells at –70C.

For transformation mix 1 mkl of ligation reaction with 20-25 mkl of competent cells. To avoid arcing during electroporation the concentration of the salt in ligation reaction should be reduced either by performing drop-dialysis or by diluting sample with water.