Preparation of electrocompetent cell and electroporation
- GYT preparation(250ml)
- 25ml glycerol (10% v/v)
- 0.31g yeast extract (0.125% w/v)
- 0.63g tryptone (0.25% w/v)
- dissolve all ingredients and filter with 0.22um pore filter
- Preparation of electrocompetent cells
- 9:10am--> innoculate 5ml each into two 95ml LB in 500ml flasks. Incubate one at 30°C and the other at 37°C with 225rpm
- 10:10am--> Measure OD: 37°C -> 0.165 x3.3=0.545/30°C-> 0.137x3.3=0.452
- 10:25am--> Measure OD: 37°C -> 0.194 x3.3=0.642 --> incubate in ice water bath
- 10:45am--> Measure OD: 30°C -> 0.208 x3.3=0.686 --> incubate in ice water bath
- centrifuge 25ml aliquots of sample(8 tubes) at 1000g, 4°C, 15min, then discard supernatant carefully and resuspend the cell pellet in 25ml of ice cold sterilized milliQ water.(volume ratio 1:1=sample aliquot:water)
- centrifuge 25ml aliquots of sample(8 tubes) at 1000g, 4°C, 20min, then discard supernatant carefully and resuspend the cell pellet in 12.5ml of ice cold sterilized milliQ water. combine two aliquot 12.5ml into 25ml(volume ratio, 2:1=sample aliquot:water)
- centrifuge 25ml aliquots of sample(4 tubes) at 1000g, 4°C, 20min, then discard supernatant carefully and resuspend the cell pellet in 1ml of ice cold 10% glycerol. combine four aliquots 1ml into 4ml in 15ml pre-cooled tubes(volume ratio, 50:1=sample aliquot:glycerol)
- centrifuge at 1000g, 4°C, 20min, then discard supernatant carefully and resuspend the cell pellet in 0.4ml of ice cold GYT medium.(volume ratio, 500:1=sample aliquot:glycerol)
- Measure OD, 2.97ml GYT medium + 0.03ml sample (100times dilution), get 0.849x100=84.9?...Wrong calculation(I thought it was 8.49), just dilute 2.5times, 40ul sample + 60ul GYT in 1.5ml microcentrifuge tube, make 40ul aliquots, store at -80c.
- Electroporation (Every steps were done aseptically in biosafety hood)
- 50ul competent cell + 0.5ul (S+B) plasmid/ 2ul S plasmid/ 2ul B plasmid each, place on the ice
- set eppendorf multiporator as 2.5KV and 5ms
- use 2mm cuvette, trasnfer cells+plasmid into cuvette, tap it slightly on to the table to make samples go down to the bottom of cuvette.
- push start button, after electroporation add 0.95ml SOC medium into cuvette right away. place a cuvette on the ice, repeat 3times
- transfer sample into chilled 1.5ml microcentrifuge tubes, incubate on ice 2min.
- incubate 3 tubes at 37°C at 250rpm for 1hr, prewarm LB+kan agar plate in 37°C incubator
- pour 200ul sample each into a agar plate, spread with L shape spreader.
- place in room temperature to make agar absorb liquid, and then incubate at 37°C (8:30pm)