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 acc 5/92
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