User:Aram Kang/Notebook/Analysis of biofilm gene expression/2008/12/26

Preparation of electrocompetent cell and electroporation

• GYT medium 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
  • store 50ml aliquot at 4°C

• 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=0.495/30°C-> 0.137x3=0.411
  • 10:25am--> Measure OD: 37°C -> 0.194 x3=0.582 --> incubate in ice water bath
  • 10:45am--> Measure OD: 30°C -> 0.208 x3=0.624 --> 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:GYT medium)
  • 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, 400ul sample + 600ul 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)

• LB + kan agar plate preparation
  • LB broth 12.5g
  • agar 10g
  • kanamycin 10mg/ml stock solution 2.5ml(working conc. 50μg/mL)