Team:UNIPV-Pavia/Measurements

From 2011.igem.org

(Difference between revisions)
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<li class="toclevel-1"><a href="#pTet_protocol"><span class="tocnumber">1</span> <span class="toctext">pTet protocol</span></a>
<li class="toclevel-1"><a href="#pTet_protocol"><span class="tocnumber">1</span> <span class="toctext">pTet protocol</span></a>
<li class="toclevel-1"><a href="#pLux_protocol"><span class="tocnumber">2</span> <span class="toctext">pLux protocol</span></a>
<li class="toclevel-1"><a href="#pLux_protocol"><span class="tocnumber">2</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 protocols</span></a>
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<li class="toclevel-1"><a href="#Enzyme"><span class="tocnumber">3</span> <span class="toctext">Enzyme activity assay</span></a>
<ul>
<ul>
<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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<li> temperature: 37°C
<li> temperature: 37°C
<li> sampling time: 5 minutes
<li> sampling time: 5 minutes
-
<li> fluorescence gain: 50
+
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements
 +
<li> fluorescence gain: 50 - 80
<li> O.D. filter: 600 nm
<li> O.D. filter: 600 nm
<li> RFP filters: 535 nm (excitation) / 620 nm (emission)
<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
 
<li> duration time: 10 - 15 hours
<li> duration time: 10 - 15 hours
</ul>
</ul>
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<li> temperature: 37°C
<li> temperature: 37°C
<li> sampling time: 5 minutes
<li> sampling time: 5 minutes
-
<li> fluorescence gain: 50
+
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements
 +
<li> fluorescence gain: 50 - 80
<li> O.D. filter: 600 nm
<li> O.D. filter: 600 nm
<li> RFP filters: 535 nm (excitation) / 620 nm (emission)
<li> RFP filters: 535 nm (excitation) / 620 nm (emission)
-
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements
 
<li> duration time: 10 - 15 hours
<li> duration time: 10 - 15 hours
</ul>
</ul>
</ol>
</ol>
</p>
</p>
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 +
 +
<a name="Enzyme"></a> <h2 class="art-postheader">
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Enzyme activity assay
 +
</h2>
 +
 +
<a name="AiiA"></a> <h2 class="art-postheader">
 +
AiiA activity
 +
</h2>
 +
 +
 +
 +
 +
 +
<div class="cleared"></div>
<div class="cleared"></div>

Revision as of 20:56, 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 1:500 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
    • 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements
    • fluorescence gain: 50 - 80
    • O.D. filter: 600 nm
    • RFP filters: 535 nm (excitation) / 620 nm (emission)
    • duration time: 10 - 15 hours

pLux 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 1:500 in 1 ml of M9 + Cm12.5 and let them grow for three hours.
  4. Induce cultures in falcon tube with 3OC6-HSL (final concentrations: 0 nM, 0.1 nM, 0.5 nM, 1 nM, 2 nM, 5 nM, 10 nM, 100 nM). 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
    • 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements
    • fluorescence gain: 50 - 80
    • O.D. filter: 600 nm
    • RFP filters: 535 nm (excitation) / 620 nm (emission)
    • duration time: 10 - 15 hours

Enzyme activity assay

AiiA activity

Laboratory for Biomedical Informatics | Centre of Tissutal Engineering

University of Pavia
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