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).

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

@@ Line 10: / Line 10: @@
 </div>
+==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
 </div>