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A small repository of synaptic protein information
Caution! In converting these protocols to HTML, some units have been scrambled.
Specifically, microliters (ul) often appear as milliliters (ml).
Caution! In converting these protocols to HTML, some units have been scrambled.
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
