Team:Valencia/protocols

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Verification protocol of resistance with LEA

• Preculture
  • Take two tubes of 25 ml and add 5 ml of LB in both of them.
  • Add 5µl of kannamicine in each tube.
  • Inoculate in each tube a colony corresponding E. coli (with LEA and without LEA).
  • Maintain the tubes during 8 hours at 37 ºC and 250 rpm.

• Culture overnight of liquid medium
  • Take two flasks of 500 ml and add 50µl of kannamicine in both flasks.
  • Inoculate 500µl of corresponding preculture.
  • Maintain the flask incubator at 37ºC and 250 rpm.

• Measure of OD of culture and dilution to exponential growth zone
  • Measure of the OD of each culture.
  • Dilution the cultures with LB fresh to OD of 0.5.
  • Grow the cultures to OD of 0.6.

• Induction with IPTG
  • Add IPTG to 1mM final concentration.
  • Incubate, both cultures, during 4 hours at 37 ºC and 250 rpm.

• Adjust of OD
  • Measure the OD of each culture.
  • Adjust the OD of both cultures at 0.4.

• Add glycerol
  • Extract three aliquots, for each culture, corresponding to the different concentrations of glycerol (the volumes are calculated for glycerol to 99,5%):
    • 0% glycerol: take 50 ml of culture and nothing of glycerol.
    • 8% glycerol: take 4 ml of glycerol and 56 % and 46 ml of culture.
    • 50% glycerol: take 25.3 ml of glycerol and 24.7 ml of culture.
  • Prepare these premixes with tubes of 50 ml (only for mixing).

• Separate the cultures for the different conditions
  • Extract twelve 1 ml aliquots from each stock of 50 ml.

• Spread to count initial colonies
  • Take 1 ml of each 50mL stock.
  • Prepare serial dilutions of each 1 ml of the samples:
    • 1/1,000
    • 1/5,000
    • 1/25,000
    • 1/125,000
  • Spread (with glass beads)LB plates (without antibiotic) with 100 µl for each dilutions.

• Stress by extremely conditions
  • Low temperatures during 2 hours and high temperatures during 45’
  • Take 1 ml of sample of each culture after each completed cycle.
  • Prepare serial dilution of each sample:
    • 1/1,000
    • 1/5,000
    • 1/25,000
    • 1/125,000