Team:Valencia/protocols
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Protocols and Methodes
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).
- Extract three aliquots, for each culture, corresponding to the different concentrations of glycerol (the volumes are calculated for glycerol to 99,5%):
- 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