Team:British Columbia/Protocols/ConsortiaFluor

From 2012.igem.org

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<h1>Consortia Fluorescence Monitoring</h1>
<h1>Consortia Fluorescence Monitoring</h1>
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1. Grow overnight 5mL cultures of auxotrophs(MetA-, TrpA-, and TyrA-)with their respective fluorescent proteins in LB media and ampicillin (100mg/mL) .  
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#Grow overnight 5mL cultures of auxotrophs(MetA<sup>-</sup>, TrpA<sup>-</sup>, and TyrA<sup>-</sup>)with their respective fluorescent proteins in LB media and ampicillin (100 mg/mL) .  
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#Spin down cells using a centrifuge (1600 g, 10 min). Pour out LB supernatant.  
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2. Spin down cells using a centrifuge (1600g, 10min). Pour out LB supernatant.  
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#Wash and resuspend in 5mL M9 media. Spin down cells using a centrifuge (1600 g, 10 min)
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#Perform step 3 three times.  
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3. Wash and resuspend in 5mL M9 media. Spin down cells using a centrifuge (1600g, 10min)
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#Resuspend in 5 mL M9 Media.
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#Measure the respective O.D 600 with a spectrometer.
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4. Perform step 3 three times.  
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#Inoculate appropriate co-culture of auxotrophs (each with a starting O.D.600 of 0.05) in a 96 well plate containing 200 μL M9 with ampicillin (100 mg/mL).
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#Incubate in Tecan plate scanner, taking measurements of O.D.600 and fluorescence outputs for time intervals of every 15 minutes for 24 hours.
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5. Resuspend in 5mL M9 Media.
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6. Measure the respective O.D 600 with a spectrometer.
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7. Inoculate appropriate co-culture of auxotrophs (each with a starting O.D.600 of 0.05) in a 96 well plate containing 200uL M9 with ampicillin (100mg/mL).
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8. Incubate in Tecan plate scanner, taking measurements of O.D.600 and fluorescence outputs for time intervals of every 15 minutes for 24 hours.
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Revision as of 01:21, 4 October 2012

British Columbia - 2012.igem.org
UBC iGEM 2012 protocols


Consortia Fluorescence Monitoring

  1. Grow overnight 5mL cultures of auxotrophs(MetA-, TrpA-, and TyrA-)with their respective fluorescent proteins in LB media and ampicillin (100 mg/mL) .
  2. Spin down cells using a centrifuge (1600 g, 10 min). Pour out LB supernatant.
  3. Wash and resuspend in 5mL M9 media. Spin down cells using a centrifuge (1600 g, 10 min)
  4. Perform step 3 three times.
  5. Resuspend in 5 mL M9 Media.
  6. Measure the respective O.D 600 with a spectrometer.
  7. Inoculate appropriate co-culture of auxotrophs (each with a starting O.D.600 of 0.05) in a 96 well plate containing 200 μL M9 with ampicillin (100 mg/mL).
  8. Incubate in Tecan plate scanner, taking measurements of O.D.600 and fluorescence outputs for time intervals of every 15 minutes for 24 hours.



Excitation and Emission Wavelengths of Fluorescent Proteins

EYFP - Excitation: 514nm Emission: 527nm

ECFP - Excitation: 439nm Emission: 476nm

ERFP - Excitation: 584nm Emission: 607nm

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