Team:UNIPV-Pavia/Measurements

From 2011.igem.org

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<li class="toclevel-1"><a href="#pTet_characterization"><span class="tocnumber">1</span> <span class="toctext">pTet characterization</span></a>
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<li class="toclevel-1"><a href="#pTet_protocol"><span class="tocnumber">1</span> <span class="toctext">pTet protocol</span></a>
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<li class="toclevel-1"><a href="#pLux_characterization"><span class="tocnumber">1</span> <span class="toctext">pLux characterization</span></a>
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<li class="toclevel-1"><a href="#pLux_protocol"><span class="tocnumber">1</span> <span class="toctext">pLux protocol</span></a>
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<li class="toclevel-1"><a href="#Enzyme"><span class="tocnumber">3</span> <span class="toctext">Enzyme activity characterization</span></a>
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<li class="toclevel-1"><a href="#Enzyme"><span class="tocnumber">3</span> <span class="toctext">Enzyme activity protocols</span></a>
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<li class="toclevel-2"><a href="#AiiA"><span class="tocnumber">3.1</span> <span class="toctext">AiiA activity</span></a></li>
<li class="toclevel-2"><a href="#AiiA"><span class="tocnumber">3.1</span> <span class="toctext">AiiA activity</span></a></li>
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pTet protocol
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<p>
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<li> Streak long term storage glycerol stocks on a LB agar plate + Cm12.5 (don't forget positive and negative controls). Let them grow over night.
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<li> Pick 3 colonies from each clone and inoculate it in 1 ml M9 + Cm12.5 in a falcon tube; let them grow over night.
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<li> Dilute cultures 1:100 in 1 ml of M9 + Cm12.5 and let them grow for three hours.
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<li> Induce cultures in falcon tube with anhydrotetracycline (aTc) (final concentrations: 0 ng/ml, 1 ng/ml, 2 ng/ml, 3 ng/ml, 4 ng/ml, 5 ng/ml, 8 ng/ml, 10 ng/ml, 50 ng/ml, 100 ng/ml). Grow cultures for three hours.
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<li> Aliquote cultures in Tecan Infinite F200 microplate reader, filling each well with 200 &mu;l of cultures. Set the automatic procedure:
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<ul>
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<li> temperature: 37°C
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<li> sampling time: 5 minutes
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<li> fluorescence gain: 50
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<li> O.D. filter: 600 nm
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<li> RFP filters: 535 nm (excitation) / 620 nm (emission)
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<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements
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<li> duration time: 10 - 15 hours
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</ul>
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</ol>
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</p>

Revision as of 20:41, 14 September 2011

UNIPV TEAM 2011

Contents

pTet protocol

  1. Streak long term storage glycerol stocks on a LB agar plate + Cm12.5 (don't forget positive and negative controls). Let them grow over night.
  2. Pick 3 colonies from each clone and inoculate it in 1 ml M9 + Cm12.5 in a falcon tube; let them grow over night.
  3. Dilute cultures 1:100 in 1 ml of M9 + Cm12.5 and let them grow for three hours.
  4. Induce cultures in falcon tube with anhydrotetracycline (aTc) (final concentrations: 0 ng/ml, 1 ng/ml, 2 ng/ml, 3 ng/ml, 4 ng/ml, 5 ng/ml, 8 ng/ml, 10 ng/ml, 50 ng/ml, 100 ng/ml). Grow cultures for three hours.
  5. Aliquote cultures in Tecan Infinite F200 microplate reader, filling each well with 200 μl of cultures. Set the automatic procedure:
    • temperature: 37°C
    • sampling time: 5 minutes
    • fluorescence gain: 50
    • O.D. filter: 600 nm
    • RFP filters: 535 nm (excitation) / 620 nm (emission)
    • 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements
    • duration time: 10 - 15 hours

Laboratory for Biomedical Informatics | Centre of Tissutal Engineering

University of Pavia
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