Preparation of Cells

1.      Inoculate 1l of L-broth with 1/100 volume of a fresh overnight culture

2.      37C, shaking, to ABS600 = 0.5-0.8

3.      Chill the flask on ice for 15-30min, centrifuge in a cold rotor 6000G, 10min

4.      Remove as much of the supernatant as possible, resuspend pellets in 1l 10% glycerol, repeat step 3

5.      Resuspend in 0.5 l 10% glycerol, repeat step 3

6.      Resuspend in 20ml ice cold 10% glycerol, repeat step 3

7.      Resuspend in 2-3ml 10% glycerol

8.      Frozen in aliquots in liquid N2, store at -80C.

Electroporation

1.      Chill electroporation cuvettes (use sterile ones, can be reused for 2-3 times) and white chamber slide

2.      Add 1-2ul plasmid/ligation product

3.      Add 40ul electroporation competent cells on the same spot where the plasmid was placed

4.      Agitate to mix, incubate for 1-2min

5.      Pulse (Setting: 1.8KV/200Ù/25uFD)

6.      Add 1ml LB in cuvette, transfer to eppendorf tube

7.      Shake for 1 hour

8.      Plate