Team:Valencia/protocols

From 2010.igem.org

(Difference between revisions)
(→Verification protocol of resistance with LEA)
 
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*Preculture
*Preculture
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**Take two tubes of 25 ml and add 5 ml of LB in both of them.
**Take two tubes of 25 ml and add 5 ml of LB in both of them.
**Add 5µl of kannamicine in each tube.
**Add 5µl of kannamicine in each tube.
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*Culture overnight of liquid medium
*Culture overnight of liquid medium
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** Take two flasks of 500 ml and add 50µl of kannamicine in both flasks.
** Take two flasks of 500 ml and add 50µl of kannamicine in both flasks.
** Inoculate 500µl of corresponding preculture.
** Inoculate 500µl of corresponding preculture.
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*Measure of OD of culture and dilution to exponential growth zone
*Measure of OD of culture and dilution to exponential growth zone
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**Measure of the OD of each culture.
**Measure of the OD of each culture.
**Dilution the cultures with LB fresh to OD of 0.5.
**Dilution the cultures with LB fresh to OD of 0.5.
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*Induction with IPTG
*Induction with IPTG
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**Add IPTG to 1mM final concentration.
**Add IPTG to 1mM final concentration.
**Incubate, both cultures, during 4 hours at 37 ºC and 250 rpm.
**Incubate, both cultures, during 4 hours at 37 ºC and 250 rpm.
*Adjust of OD
*Adjust of OD
-
 
**Measure the OD of each culture.
**Measure the OD of each culture.
**Adjust the OD of both cultures at 0.4.
**Adjust the OD of both cultures at 0.4.
*Add glycerol
*Add glycerol
-
 
**Extract three aliquots, for each culture, corresponding to the different concentrations of glycerol (the volumes are calculated for glycerol to 99,5%):
**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.
***0% glycerol: take 50 ml of culture and nothing of glycerol.
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*Separate the cultures for the different conditions
*Separate the cultures for the different conditions
-
 
**Extract twelve 1 ml aliquots from each stock of 50 ml.
**Extract twelve 1 ml aliquots from each stock of 50 ml.
*Spread to count initial colonies
*Spread to count initial colonies
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**Take 1 ml of each 50mL stock.
**Take 1 ml of each 50mL stock.
**Prepare serial dilutions of each 1 ml of the samples:
**Prepare serial dilutions of each 1 ml of the samples:
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*Stress by extremely conditions
*Stress by extremely conditions
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**Low temperatures during 2 hours and high temperatures during 45’
**Low temperatures during 2 hours and high temperatures during 45’
**Take 1 ml of sample of each culture after each completed cycle.
**Take 1 ml of sample of each culture after each completed cycle.

Latest revision as of 01:52, 28 October 2010


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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
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