TRANSFORMATION OF E. Coli BY ELECTROPORATION
(BIORAD)
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' @ 4oC.
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 @ -70oC. 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) @ 37oC 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
acc 5/92
上一篇：Preparation of Competent Cells (from PMB) 下一篇：Transformation of Compenent E. coli[Yale University]
