Difference between revisions of "Team:Tuebingen/Experiments"

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{{Tuebingen}}
 
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<html>
 
  
  
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<script type="text/javascript">
 
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dias = ['Cloning', 'Buffers and Media', 'Assays and further methods'];
 
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</script>
 
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<div id="dia0" class="dia">
 
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<label class="collapse" for="_1"><h4>> 3A-Assembly</h4></label>
 
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  <input id="_1" type="checkbox">
 
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  <div><ul>
 
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  <li>1&mu;l 10x ligase buffer</li>
 
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  <li>0,5&mu;l ligase</li>
 
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  <li>1&mu;l ATP</li>
 
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  <li>0,51&mu;l linearised vector</li>
 
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  <li>33,5&mu;l insert 1</li>
 
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  <li>33,5&mu;l insert 2</li>
 
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  </ul>
 
  
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  <p>Method:</p>
 
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  <ul>
 
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  <li>Mix all components</li>
 
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  <li>Incubate overnight at 4&deg;C </li>
 
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  <li>Gelpurify using agarose gels</li>
 
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  </ul></div>
 
  
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<label class="collapse" for="_2"><h4>> Chemically Competent E.coli Cells</h4></label>
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<label class="collapse" for="_27"><h4>> Agarose Gels</h4></label>
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  <input id="_2" type="checkbox">
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  <div><ul>
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  <li>autoclave 250mL LB in Erlenmeyer beaker</li>
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  <li>plate cells from cell stock on agarose plate with appropriate antibiotics</li>
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  <li>inoculate one clone in 5mL of LB with antibiotics, grow at 37&deg;C o/n</li>
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  <li>expand to 500mL and grow until OD600nm = 0.35</li>
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  <li>transfer into 50mL Falcon tubes</li>
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  <li>refrigerate centrifuge at 4&deg;C, chill 2x 25mL 0.1M CaCl2 (Falcon tubes) and 5mL 0.1M CaCl2 with 10%
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Glycerol</li>
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  <li>spin down cells at 2000g for 10Min at 4&deg;C</li>
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  <li>resuspend 5 Falcon cell pellets in 25mL ice cold 0.1M CaCl2</li>
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  <li>spin down at 2000g for 10Min at 4&deg;C</li>
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  <li>resuspend pellet in 2x5mL (=10mL) ice cold 0.1M CaCl2 with 10% Glycerol</li>
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  <li>freeze 50-100&mu;l aliquots</li>
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  <li>store at 20&deg;C</li>
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  </ul></div>
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  <label class="collapse" for="_3"><h4>> ColonyPCR</h4></label>
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  <input id="_3" type="checkbox">
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  <div>
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  <ul>
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  <li>0,1&mu;l forward primer (10&mu;M)</li>
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  <li>0,1&mu;l reverse primer (10&mu;M)</li>
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  <li>5&mu;l 2x Q5 Mastermix</li>
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  <li>4,8&mu;l H2O</li>
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  </ul>
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Method:
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<ul>
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<li>Pick a colony from plate and streak in a PCR tube. Mix components and add to tube. </li><li>Run PCR:</li>
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<li>95&deg;C 5:00min</li>
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<li>95&deg;C 0:45min</li>
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<li>53&deg;C 0:45min</li>
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<li>72&deg;C 1:30min</li>
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<li>72&deg;C 10:00min</li>
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<li>4&deg;C inf</li>
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</ul></div>
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  <label class="collapse" for="_4"><h4>> DoubleDigest Restriction of gBlocks and BioBricks</h4></label>
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  <input id="_4" type="checkbox">
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  <div><ul>
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<li>2&mu;l 10x buffer (corresponding to enzymes)</li>
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<li>0,5&mu;l restriction enzyme 1</li>
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<li>0,5&mu;l restriction enzyme 2</li>
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<li>1&mu;g DNA </li>
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<li>fill up to 20&mu;l with H2O </li>
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</ul>
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<p>Method:</p>
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<ul>
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<li> mix all components</li>
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<li> incubate at 37&deg;C for 2h</li>
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<li> heatinactivate enzymes at 80&deg;C for 10min or use for gel purification</li>
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</ul></div>
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  <label class="collapse" for="_5"><h4>> Gelpurification</h4></label>
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  <input id="_5" type="checkbox">
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  <div><ul>
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<p>This is a modified protocol for the Wizard SV Gel and PCR CleanUp System Kit from Promega</p>
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<li> Following electrophoresis, excise DNA band from gel and place gel slice in a 1,5ml microcentrifuge tube</li>
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<li> Add 10&mu;l Membrane Binding Solution per 10mg of gel slice. Vortex and incubate at 5060&deg;C until gel slice is
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completely dissolved</li>
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<li> Insert SV Minicolumn into Collection Tube</li>
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<li> Transfer dissolved gel mixture to the Minicolumn assembly. Incubate at room temp. for 1 minute</li>
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<li> Centrifuge at 16,000xg for 1 min. Discard flowthrough and reinsert Minicolumn into Collection tube</li>
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<li> Add 700&mu;l Membrane Wash Solution (ethanol added). Centrifuge at 16,000xg for 1 min</li>
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<li> Discard flowthrough and reinsert Minicolumn into Collection tube</li>
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<li> Repeat this step with 500&mu;l Membrane Wash Solution. Centrifuge at 16,000xg for 5 min</li>
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<li> Empty the Collection Tube and centrifuge the column assembly for 1,5 min</li>
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<li> Leave the tubes open for 10 min (to let any rest of ethanol evaporate)</li>
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<li> Carefully transfer the Minicolumn to a clean 1.5ml microcentrifuge tube</li>
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<li> Add 30&mu;l of NucleaseFreeWater (65&deg;C) to the Minicolumn. Incubate at room temperature for 10 min</li>
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<li> Centrifuge at 16,000xg for 1 min.</li>
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<li> Incubate sample at 65&deg;C for 5min</li>
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<li> Discard Minicolumn and store DNA at 4&deg;C or 20&deg;C</li>
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</ul></div>
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  <label class="collapse" for="_6"><h4>> Ligation</h4></label>
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  <input id="_6" type="checkbox">
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  <div><ul>
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<li>1&mu;l 10x ligase buffer</li>
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<li>0,5&mu;l ligase</li>
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<li>1&mu;l ATP</li>
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<li>0,51&mu;l linearised vector</li>
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<li>7&mu;l insert</li>
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</ul>
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<p>Method:</p>
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<ul>
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<li>Mix all components</li>
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<li>Incubate overnight at 4&deg;C</li>
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<li>Gelpurify using agarose gels</li>
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</ul>
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<h4>MutagenesisPCR</h4>
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<ul>
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<li>200ng template </li>
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<li>1&mu;l forward primer (2mM)</li>
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<li>1&mu;l reverse primer (2mM)</li>
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<li>1&mu;l Phu polymerase</li>
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<li>1&mu;l dNTPs</li>
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<li>10&mu;l 5x Buffer</li>
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<li>to 50&mu;l with H2O</li>
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<li>Mix components</li>
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<li>Run thermocycler:</li>
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<li>98&deg;C 0:30min</li>
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<li>98&deg;C 0:45min</li>
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<li>55&deg;C 0:45min</li>
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<li>72&deg;C 7:00 min</li>
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<li>72&deg;C 1:00 min</li>
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<li>4&deg;C inf </li>
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</ul>
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<h4>Oligo Annealing</h4>
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<ul>
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<li>2&mu;l oligo 1</li>
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<li>2&mu;l oligo 2</li>
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<li>96&mu;l elution buffer</li>
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</ul>
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<p>Method:</p>
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<ul>
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<li>Mix all components</li>
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<li>incubate for 10 minutes at 95&deg;C</li>
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<li>take tube with heat block out of heat and let it cool down to room temperature</li>
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</ul></div>
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  <label class="collapse" for="_7"><h4>> Smallscale plasmid preparation</h4></label>
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  <input id="_7" type="checkbox">
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  <div><p>Preparation was done using the PureYield™ Plasmid Miniprep System by Promega´</p>
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<ul>
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<li>Pick colonies for overnight cultures in 23ml LB medium with antibiotic
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grow overnight</li>
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<li>Spin down 1,5ml bacteria at maximum speed for 30 minutes, discard supernatant and add further 1,5ml, spin again and
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discard supernatant</li>
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<li>Resuspend the bacteria pellet in 600&mu;l H2O</li>
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<li>Add 100 &mu;l Cell Lysis Buffer, invert six times</li>
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<li>Add 350 &mu;l cold Neutralization Solution, mix thoroughly by inverting</li>
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<li>Centrifuge 30s at maximum speed</li>
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<li>Transfer supernatant to PureYield Minicolumn in collection tube</li>
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<li>Centrifuge for 30 sec, discard the flow-through </li>
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<li>Add 200 &mu;l Endotoxin Removal Wash(ERB), centrifuge for 30sec, discard the flow-through.</li>
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<li>Add 400 &mu;l Column Wash Solution(CWC), centrifuge for 30sec, discard the flow-through.</li>
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<li>Place column in eppendorf tube, add 30 &mu;l Elution Buffer to minicolumn matrix, incubate for 1 min</li>
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<li>Centrifuge column for 30s at maximum speed, flow-through contains plasmid DNA</li>
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<li>Measure the DNA concentration using a Nanodrop</li>
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</ul></div>
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  <label class="collapse" for="_8"><h4>> Testrestriction</h4></label>
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  <input id="_8" type="checkbox">
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  <div><ul>
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<li>0,25&mu;l Enzyme 1</li>
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<li>0,25&mu;l Enzyme 2</li>
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<li>1&mu;l 10x buffer (corresponding to enzymes)</li>
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<li>200-300ng DNA</li>
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<li>Fill up with water to 10&mu;l.</li>
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</ul>
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<p>Method:</p>
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<ul>
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<li>Incubate at 37&deg;C for 1h</li>
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<li>Run on 1% agarose gel</li>
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</ul>
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</div>
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<label class="collapse" for="_9"><h4>> Transformation of Bacteria</h4></label>
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  <input id="_9" type="checkbox">
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  <div><ul>
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<li>50&mu;l chemically competent E. coli </li>
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<li>35&mu;l overnight ligation mix OR 1ng purified plasmid DNA</li>
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</ul>
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<p>Method:</p>
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<ul>
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<li> Thaw bacteria pellet on ice</li>
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<li> add ligation mixture or purified plasmid to bacteria</li>
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<li> incubate for 20min on ice</li>
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<li> heat shock bacteria for 45s at 42&deg;C</li>
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<li> incubate bacteria on ice for 2min</li>
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<li> add 700&mu;l LB medium without antibiotics</li>
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<li> incubate for 1h at 37&deg;C</li>
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<li> spin down bacteria at 2g for 2min</li>
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<li> discard supernatant by tipping the tube</li>
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<li> resuspend bacterial pellet in leftover supernatant (50100&mu;l)</li>
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<li> streak bacteria onto LBagar plates with antibiotics and incubate overnight </li>
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</ul></div>
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<label class="collapse" for="_10"><h4>> Transformation of Yeast</h4></label>
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  <input id="_10" type="checkbox">
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  <div><ul>
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<li>ca. 10ml YPD</li>
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<li>100&mu;l OneStep buffer</li>
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<li>20&mu;g ssDNA</li>
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<li>100-500 ng plasmid DNA</li>
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<li>fresh YPD selective plate</li>
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</ul>
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<p>Method:</p>
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<ul>
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<li> Scrape some yeast cells off a fresh YPD plate and inoculate in liquid YPD (= culture).</li>
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<li> Thaw ssDNA (e.g. salmon sperm DNA) and heat for 10 min at 95 &deg;C, then chill on ice.</li>
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<li> Pellet 1 mL of culture by centrifugation at > 13.000 rpm.</li>
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<li> Discard supernatant and resuspend cells in 100 &mu;L OneStep buffer, vortex heavily</li>
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<li> Add 20 &mu;g ssDNA (10 &mu;L of 2 mg/mL) and 100  500 ng plasmid DNA to be transformed</li>
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<li> Vortex and incubate at 45 &deg;C for 2 h</li>
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<li> Add 1 mL YPD, mix and spin 10 sec at full speed, discard supernatant</li>
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<li> Resuspend cell pellet in 1000 &mu;L YPD and plate 100 &mu;L directly on appropriate selective plates</li>
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<li> Colonies appear after 2 days of incubation at 30 &deg;C</li>
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</ul></div>
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</div>
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<div id="dia1" class="dia">
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<label class="collapse" for="_11"><h4>> TBE </h4></label>
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  <input id="_11" type="checkbox">
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  <div><ul>
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  <li>1M tris</li>
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  <li>0,85M B(OH)3</li>
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  <li>2mM EDTA</li>
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  <li>pH=8,0</li>
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  </ul></div>
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<label class="collapse" for="_12"><h4>> SDS-PAGE running buffer</h4></label>
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  <input id="_12" type="checkbox">
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  <div><ul>
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<li>20mM Tris/HCl pH 7,6</li>
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<li>250mM Glycin</li>
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<li>0,1% (w/v) SDS</li>
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<li>in ddH2O</li>
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</ul></div>
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<label class="collapse" for="_13"><h4>> 1x Laemmli</h4></label>
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  <input id="_13" type="checkbox">
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  <div><ul>
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<li>50mM Tris pH 6,8</li>
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<li>1,25mM EDTA pH 8</li>
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<li>12,5% (v/v) Glycin</li>
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<li>2% (w/v) SDS</li>
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<li>50mM DTT</li>
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<li>2,5% (v/v) -Mercaptoethanol</li>
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<li>0,025% (w/v) Bromphenolblau</li>
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<li>in ddH2O</li>
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</ul></div>
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<label class="collapse" for="_14"><h4>> LB</h4></label>
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  <input id="_14" type="checkbox">
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  <div>
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<ul>
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<li>20g Lennox Broth</li>
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<li>to 1l with H2O</li>
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</ul></div>
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<label class="collapse" for="_15"><h4>> LB/Agar</h4></label>
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  <input id="_15" type="checkbox">
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  <div>
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<ul>
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<li>35g LB-Agar (Lennox)</li>
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<li>to 1l H2O</li>
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</ul></div>
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<label class="collapse" for="_16"><h4>> SC-media</h4></label>
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  <input id="_16" type="checkbox">
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  <div><ul>
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<li>2g yeast nitrogen base w/o amino acids</li>
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<li>0,25g synthetic complete drop-out mix</li>
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<li>16,5mg adenine-sulfate</li>
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<li>to 300ml H2O</li>
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<li>adjust pH to 5.6</li>
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<li>add agar if needed </li>
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</ul></div>
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<label class="collapse" for="_17"><h4>> synthetic complete drop-out mix</h4></label>
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  <input id="_17" type="checkbox">
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  <div><ul>
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<li>1g adenine hemisulfate</li>
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<li>1g arginine-HCl</li>
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<li>1g histidine HCl*</li>
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<li>1g isoleucine</li>
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<li>2g leucine*</li>
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<li>2g lysine-HCl</li>
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<li>2g methionine*</li>
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<li>1,5g phenylalanine</li>
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<li>1g serine</li>
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<li>1g threonine</li>
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−
<li>1,5g tryptophane*</li>
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−
<li>1g tyrosine</li>
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<li>0,6g uracil*</li>
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<li>4,5g valine</li>
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<li>for dropout mix, omit appropriate components, labelled with *</li>
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<li>combine ingredients and mix thoroughly</li>
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</ul>
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</div>
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<label class="collapse" for="_18"><h4>> YEP (yeast extract peptone)</h4></label>
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  <input id="_18" type="checkbox">
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  <div><ul>
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<li>3g yeast extract</li>
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<li>6g peptone</li>
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<li>30mg adenine hemisulphate</li>
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<li>300ml H2O</li>
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<li>if needed, add 5g agar</li>
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</ul></div>
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<label class="collapse" for="_19"><h4>> 20% Glucose/Galactose/raffinose</h4></label>
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  <input id="_19" type="checkbox">
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  <div>
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<ul>
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<li>20g of appropriate sugar</li>
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<li>100ml H2O</li>
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</ul>
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</div>
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<label class="collapse" for="_20"><h4>> antibiotics concentration</h4></label>
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  <input id="_20" type="checkbox">
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  <div><ul>
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<li>CA 34µg/ml</li>
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<li>Amp 100µg/ml</li>
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</ul></div>
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<label class="collapse" for="_21"><h4>> One-step buffer</h4></label>
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  <input id="_21" type="checkbox">
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  <div><ul>
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<li>0,2M LiAc</li>
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<li>40% PEG4000</li>
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<li>100mM DTT</li>
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<li>sterile filtrated</li>
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</ul></div>
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<h2>SDS-Gels</h2>
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  <label class="collapse" for="_22"><h4>> 12% separation gel</h4></label>
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  <input id="_22" type="checkbox">
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  <div><ul>
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<li>40,5% acrylamide (30%)</li>
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<li>0,375M Tris (pH 8,8)</li>
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<li>1% SDS (10%)</li>
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<li>1% APS(10%)</li>
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<li>0,1% TEMED</li>
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</ul></div>
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  <label class="collapse" for="_23"><h4>> 5% stacking gel</h4></label>
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  <input id="_23" type="checkbox">
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  <div><ul>
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<li>17% acrylamide (30%)</li>
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<li>0,125M Tris (pH 6,8)</li>
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<li>1% SDS (10%)</li>
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<li>1% APS (10%)</li>
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<li>0,1% TEMED</li>
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</ul></div>
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<h2>Ni-NTA buffers</h2>
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<p>All buffers are from the Qiagen "Ni-NTA Agarose Purification of 6xHis-tagged Proteins from E. coli under Native
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Conditions - Quick-Start"-protocol.</p>
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  <label class="collapse" for="_24"><h4>> Lysis buffer (pH = 8)</h4></label>
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  <input id="_24" type="checkbox">
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  <div><ul> 
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<li>50 mM NaH2PO4 </li>
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<li>300 mM NaCl </li>
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<li>10 mM Imidazole</li>
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</ul> </div>
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  <label class="collapse" for="_25"><h4>> Washing buffer (pH = 8)</h4></label>
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  <input id="_25" type="checkbox">
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  <div><ul>
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<li> 50 mM NaH2PO4 </li>
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<li>300 mM NaCl </li>
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<li>20 mM Imidazole</li>
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</ul></div>
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  <label class="collapse" for="_26"><h4>> Elution buffer (pH = 8)</h4></label>
+
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  <input id="_26" type="checkbox">
+
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  <div><ul>
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<li>50 mM NaH2PO4</li>
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<li>300 mM NaCl</li>
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<li>250 mM Imidazole</li>
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</ul></div>
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<h2>Experiments</h2>
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<li>Cast gels with composition as described <here> (Link bitte einfügen zu der Zusammensetzung)</li>
 
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<li>Load samples with SDS-PAGE loading dye </li>
 
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<li>Run gels at 150V until front is run through the gel</li></ul></div>
 
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<li>Fluorescence was measured with Tecan plate reader (Infinite M200) and analysed with Tecan i-control v1.10</li>
 
<li>Fluorescence was measured with Tecan plate reader (Infinite M200) and analysed with Tecan i-control v1.10</li>
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<label class="collapse" for="_30"><h4>> Fluorescent-peptide-coupling Assay</h4></label>
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<li> incubate 100 µl of Ni-NTA purified eluate with 10 µl (10 mg/ml, 7,90 mM) peptide solution for 24 or 48 hours at 5°C or room temperature</li>
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<li> dilute with 110 µl of 2x Laemmli buffer</li>
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<li> incubate at 95°C for 10 minutes</li>
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<li> load onto SDS polyacrylamide gel</li></ul></div>
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<label class="collapse" for="_31"><h4>> Fluorescence Microscopy</h4></label>
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<li>We used a Zeiss LSM 710 microscope with a Plan-Apochromat 63x/1.40 Oil DIC M27 objective. All pictures were taken  
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<li>We used a Zeiss LSM 710 microscope with a Plan-Apochromat 63x/1.40 Oil DIC M27 objective. All pictures were taken with one active fluorescent channel and brightfield channel. Dronpa was observed by using a 514 nm laser (2% power) for excitation, and RFP was observed by using a 633 nm laser (2% power) for excitation</li>
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with one active fluorescent channel and brightfield channel. Dronpa was observed by using a 514 nm laser (2% power) for  
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excitation, and RFP was observed by using a 633 nm laser (2% power) for excitation</li>
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<li>Illumination of dronpa was performed in vivo using a 488 nm argon laser at 50% power for off switching and a 405 nm  
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<li>Illumination of dronpa was performed in vivo using a 488 nm argon laser at 50% power for off switching and a 405 nm for on switching of fluorescence. The illumination of cells was either performed using continuous illumination for 10 to 20 seconds or by performing a line scan with the illumination laser.</li></ul></div>
  
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for on switching of fluorescence. The illumination of cells was either performed using continuous illumination for 10 to
 
  
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<li>Grow overnight culture of yeast:Induction or repression of luciferase expression by addition of different  
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galactose/glucose/raffinose concentrations for pGal or pSUC promoters.</li>
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<li>NanoLuc luciferase protocol: add 25 µl cell culture, 25 µl reconstituted luciferase assay reagent and 50 µl PBS in 96 well plate (white, clear bottom)</li>
  
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<li>NanoLuc luciferase protocol: add 25 µl cell culture, 25 µl reconstituted luciferase assay reagent and 50 µl PBS in 96
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<li>Firefly luciferase protocol: mix either cell suspension or cell lysate 1:1 with reconstituted luciferase assay reagent</li>
  
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well plate (white, clear bottom)</li>
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<li>Luciferase activity measured with Tecan plate reader (Infinite M200) and analysed with Tecan i-control v1.10. </li></ul></div>
  
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<li>Firefly luciferase protocol: mix either cell suspension or cell lysate 1:1 with reconstituted luciferase assay
 
  
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reagent</li>
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<label class="collapse" for="_34"><h4>> MALDI-TOF mass spectrometry</h4></label>
  
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<li> incubate 100 µl of Ni-NTA purified eluate with 10 µl (10 mg/ml, 7,90 mM) peptide solution for 24 or 48 hours at 5°C
 
  
−
or room temperature</li>
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<li>Cast gels with composition as described <here> (Link bitte einfügen zu der Zusammensetzung)</li>
  
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<li> dilute with 110 µl of 2x Laemmli buffer</li>
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<li>Load samples with SDS-PAGE loading dye </li>
  
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<li> incubate at 95°C for 10 minutes</li>
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<li>Run gels at 150V until front is run through the gel</li></ul></div>
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Revision as of 23:14, 18 September 2015



<label class="collapse" for="_27">

> Agarose Gels

</label> 
 <input id="_27" type="checkbox">


  • cast gels
  • load samples with loading dye
  • run gels at 120 V


<label class="collapse" for="_28">

> Expression

</label> 
 <input id="_28" type="checkbox">


  • pETue-NAGA-Intein was transformed into E.coli BL21(DE3)
  • grow culture to OD_600 = 0.3
  • induce with 0.2 mM IPTG
  • express O/N at room temperature
  • centrifuge at 6000 g for 30 minutes at 4°C
  • resuspend pellet in 10 ml lysis buffer
  • sonicate for a total of 3 minutes (40% amplitude, 1 s intervals)


<label class="collapse" for="_29">

> Fluorescence assay

</label> 
 <input id="_29" type="checkbox">


  • Cells were measured undiluted or in 1:2 dilution in 96 well plates (black, flat bottom)
  • Following excitation and emission wavelengths were used for the different fluorescent proteins:
  • GFP
    • Exc: 488 nm
    • Em: 520 nm
    • Dronpa
      • Exc: 503 nm
      • Em: 535 nm
      • RFP
        • Exc: 584 nm
        • Em: 616 nm
        • Fluorescence was measured with Tecan plate reader (Infinite M200) and analysed with Tecan i-control v1.10
          • <label class="collapse" for="_30">

          > Fluorescent-peptide-coupling Assay

          </label> 
           <input id="_30" type="checkbox">


          • incubate 100 µl of Ni-NTA purified eluate with 10 µl (10 mg/ml, 7,90 mM) peptide solution for 24 or 48 hours at 5°C or room temperature
          • dilute with 110 µl of 2x Laemmli buffer
          • incubate at 95°C for 10 minutes
          • load onto SDS polyacrylamide gel


          <label class="collapse" for="_31">

          > Fluorescence Microscopy

          </label> 
           <input id="_31" type="checkbox">


            Sample preparation


          • A thin layer of low melting agarose was supplied on glass slides
          • 10 µl Yeast cells grown overnight were inoculated in the solidified agarose
          • samples were used for microscopy after at least 30min incubation at 30° in a humid environment


            • Microscopy Setup


          • We used a Zeiss LSM 710 microscope with a Plan-Apochromat 63x/1.40 Oil DIC M27 objective. All pictures were taken with one active fluorescent channel and brightfield channel. Dronpa was observed by using a 514 nm laser (2% power) for excitation, and RFP was observed by using a 633 nm laser (2% power) for excitation


            • Dronpa Illumination


          • Illumination of dronpa was performed in vivo using a 488 nm argon laser at 50% power for off switching and a 405 nm for on switching of fluorescence. The illumination of cells was either performed using continuous illumination for 10 to 20 seconds or by performing a line scan with the illumination laser.


          <label class="collapse" for="_32">

          > Ni-NTA Purification

          </label> 
           <input id="_32" type="checkbox">


            Pellet

          • centrifugation of the bacterial culture for 10 min at 8000 rpm at 4 °C
          • discard the supernatant
            • Lysis
          • per 1g wet weight of the pellet add 4ml lysis buffer
          • fill up to 0.5 mg/ml with lysozyme
          • incubation on ice for 2 h
          • centrifuge for 30 min at 10000 g at 4 °C
            • Column
          • equilibrate the column with 0.5 ml Ni-NTA (0.25 ml bed volume)
          • add 0.5 ml lysis buffer and slightly centrifuge the buffer through the column, it has to stay wet (do twice)
          • pump the cell lysate (supernatant) for 1 h at ice through the column
            • Wash
          • twice with 1.25ml wash buffer
          • four times with 0.5 ml elution buffer
          • recover the supernatant


          • Load a sample of every step in an SDS gel.


          <label class="collapse" for="_33">

          > Luciferase Assay

          </label> 
           <input id="_33" type="checkbox">


          • Grow overnight culture of yeast:Induction or repression of luciferase expression by addition of different galactose/glucose/raffinose concentrations for pGal or pSUC promoters.
          • NanoLuc luciferase protocol: add 25 µl cell culture, 25 µl reconstituted luciferase assay reagent and 50 µl PBS in 96 well plate (white, clear bottom)
          • Firefly luciferase protocol: mix either cell suspension or cell lysate 1:1 with reconstituted luciferase assay reagent
          • Luciferase activity measured with Tecan plate reader (Infinite M200) and analysed with Tecan i-control v1.10.


          <label class="collapse" for="_34">

          > MALDI-TOF mass spectrometry

          </label> 
           <input id="_34" type="checkbox">


            Preparation and trypsin digest:

          • cut bands from SDS polyacrylamide gel
          • cover gel piece with Buffer2 for 30 min to discolor
          • cover with acetonitrile (ACN) for 10 min to dehydrate
          • cover with Buffer1 containing 10 mM DTT to reduce for 45 min
          • cover with Buffer1 containing 55 mM iodoacetamide (IAA) to alkylate for 30 min
          • cover with Buffer1 for 15 min to wash
          • cover with Buffer2 for 10 min to wash
          • cover with 100% ACN for 10 min to dehydrate
          • cover with 20 µl Buffer1 containing 100 ng Trypsin
          • digest for 5 h at 37 °C
          • stop reaction by addition of 2 µl of 10% TFA
          • extract peptides by incubation for 30 minutes


            • Buffer1: 50 mM ammoniumbicarbonat (AB) Buffer2: 70% 50 mM AB, 30% ACN


            Measurement:

          • mix 1 ml of sample with 1 µl of gentisic acid matrix solution
          • pipette unto target
          • wait for crystallisation
          • measure with MALDI-TOF
          • analyse data using FlexAnalysis und FlexControl


            • The result score is calculated as Score = -log10(P).



          <label class="collapse" for="_35">

          > Purification

          </label> 
           <input id="_35" type="checkbox">


          • centrifuge lysate at 15,000 g for 30 min
          • purify supernatant with Ni-NTA beads (native conditions, gravity flow)
          • elute once with 500 µl elution buffer


          <label class="collapse" for="_36">

          > SDS-page

          </label> 
           <input id="_36" type="checkbox">


          • Cast gels with composition as described <here> (Link bitte einfügen zu der Zusammensetzung)
          • Load samples with SDS-PAGE loading dye
          • Run gels at 150V until front is run through the gel