Transformation of E. coli by Electroporation [Stanford University]

Preparation of electroporation cells

1. Prepare an overnight of NM522 in minimal medium.

2. Inoculate 1L LB with 10ml (1/100th vol) of the overnight and grow to A600 = 0.5- 1.0.

3. Pellet cells 5krpm, 15' @ 4℃.

4. Wash cells in 1L ice-cold water and pellet again.

5. Wash cells in 500ml ice-cold water and pellet again.

6. Wash cells in 20ml ice-cold 10% glycerol (in water) and pellet.

7. Resuspend cells in a final volume of 2-3ml 10% glycerol (‰1010cells/ml).

8. Freeze in 40l aliquots on dry ice and store @ -70℃. When testing the efficiency, transform with 1ng supercolied plasmid and plate out 50l to get 200-300 colonies.

Electroporation

1. Chill cuvette (0.2cm gap) on ice.

2. Mix <4l ligation mix with an aliquot of cells.

3. Transfer cells+DNA to the bottom of the cuvette- avoid forming bubbles.

4. Electroporate at 200ohms, 25mfarads and 2.5kvolts. The time constant should be in the 3-5msec range.

5. Immediately add 1ml SOC medium and transfer to a culture tube.

6. Shake (slower than usual, 225rpm is better) @ 37℃ for 30-60 min.

7. Plate out on selection medium.

SOC Medium

1L:

2% Bactotryptone 20g

0.5% Bactoyeast extract 5g

10mM NaCl 2.0ml 5M NaCl

2.5mM KCl 2.5ml 1M KCl

10mM MgCl₂ 10.0ml 1M MgCl₂

10mM MgSO₄ 10.0ml 1M MgSO₄

20mM Glucose (‰0.2%) 10.0ml 20% Glucose

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