Difference between revisions of "Team:Tuebingen/Methods"

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</script>
 
</script>
  
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<div id="dia0" class="dia">
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<div class="contentArea">
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<br>
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<div class="contentRow">
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<label class="collapse" for="_1"><h4>> 3A-Assembly</h4></label>
+
<h1>Methods</h1>
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  <input id="_1" type="checkbox">  
+
</div>
−
  <div><ul>
+
<div class="contentRow">
−
  <li>1&mu;l 10x ligase buffer</li>
+
<div class="contentCell contentSmallCell">
−
  <li>0,5&mu;l ligase</li>
+
Chemically Competent <i>E. coli</i> Cells
−
  <li>1&mu;l ATP</li>
+
</div>
−
  <li>0,51&mu;l linearised vector</li>
+
<div class="contentCell">
−
  <li>33,5&mu;l insert 1</li>
+
<li>autoclave 250mL LB in Erlenmeyer beaker</li>
−
  <li>33,5&mu;l insert 2</li>
+
<li>plate cells from cell stock on agarose plate with appropriate antibiotics</li>
−
  </ul>
+
<li>inoculate one clone in 5mL of LB with antibiotics, grow at 37&#176;C o/n</li>
−
 
+
<li>expand to 500mL and grow until OD600nm = 0.35</li>
−
  <p>Method:</p>
+
<li>transfer into 50mL Falcon tubes</li>
−
  <ul>
+
<li>refrigerate centrifuge at 4&#176;C, chill 2x 25mL 0.1M CaCl2 (Falcon tubes) and 5mL 0.1M CaCl2 with 10% Glycerol</li>
−
  <li>Mix all components</li>
+
<li>spin down cells at 2000g for 10Min at 4&#176;C</li>
−
  <li>Incubate overnight at 4&deg;C </li>
+
<li>resuspend 5 Falcon cell pellets in 25mL ice cold 0.1M CaCl2</li>
−
  <li>Gelpurify using agarose gels</li>
+
<li>spin down at 2000g for 10Min at 4&#176;C</li>
−
  </ul></div>
+
<li>resuspend pellet in 2x5mL (=10mL) ice cold 0.1M CaCl2 with 10% Glycerol</li>
−
 
+
<li>freeze 50-100&#956;l aliquots</li>
−
<label class="collapse" for="_2"><h4>> Chemically Competent <i>E. coli</i> Cells</h4></label>
+
<li>store at 20&#176;C</li>  
−
  <input id="_2" type="checkbox">
+
</div>
−
  <div><ul>
+
</div>
−
  <li>autoclave 250mL LB in Erlenmeyer beaker</li>
+
<div class="contentRow">
−
  <li>plate cells from cell stock on agarose plate with appropriate antibiotics</li>
+
<div class="contentCell contentSmallCell">
−
  <li>inoculate one clone in 5mL of LB with antibiotics, grow at 37&deg;C o/n</li>
+
DoubleDigest Restriction of gBlocks and BioBricks
−
  <li>expand to 500mL and grow until OD600nm = 0.35</li>
+
</div>
−
  <li>transfer into 50mL Falcon tubes</li>
+
<div class="contentCell">
−
  <li>refrigerate centrifuge at 4&deg;C, chill 2x 25mL 0.1M CaCl2 (Falcon tubes) and 5mL 0.1M CaCl2 with 10%  
+
<li>2&#956;l 10x buffer (corresponding to enzymes)</li>
−
 
+
<li>0,5&#956;l restriction enzyme 1</li>
−
Glycerol</li>
+
<li>0,5&#956;l restriction enzyme 2</li>
−
  <li>spin down cells at 2000g for 10Min at 4&deg;C</li>
+
<li>1&#956;g DNA </li>
−
  <li>resuspend 5 Falcon cell pellets in 25mL ice cold 0.1M CaCl2</li>
+
<li>fill up to 20&#956;l with H2O </li>
−
  <li>spin down at 2000g for 10Min at 4&deg;C</li>
+
−
  <li>resuspend pellet in 2x5mL (=10mL) ice cold 0.1M CaCl2 with 10% Glycerol</li>
+
−
  <li>freeze 50-100&mu;l aliquots</li>
+
−
  <li>store at 20&deg;C</li>
+
−
  </ul></div>
+
−
 
+
−
 
+
−
  <label class="collapse" for="_3"><h4>> ColonyPCR</h4></label>
+
−
  <input id="_3" type="checkbox">
+
−
  <div>
+
−
  <ul>
+
−
  <li>0,1&mu;l forward primer (10&mu;M)</li>
+
−
  <li>0,1&mu;l reverse primer (10&mu;M)</li>
+
−
  <li>5&mu;l 2x Q5 Mastermix</li>
+
−
  <li>4,8&mu;l H2O</li>
+
−
  </ul>
+
−
 
+
−
Method:
+
−
<ul>
+
−
<li>Pick a colony from plate and streak in a PCR tube. Mix components and add to tube. </li><li>Run PCR:</li>
+
−
<li>95&deg;C 5:00min</li>
+
−
<li>95&deg;C 0:45min</li>
+
−
<li>53&deg;C 0:45min</li>
+
−
<li>72&deg;C 1:30min</li>
+
−
<li>72&deg;C 10:00min</li>
+
−
<li>4&deg;C inf</li>
+
−
</ul></div>
+
−
 
+
−
 
+
−
 
+
−
  <label class="collapse" for="_4"><h4>> DoubleDigest Restriction of gBlocks and BioBricks</h4></label>
+
−
  <input id="_4" type="checkbox">
+
−
  <div><ul>
+
−
<li>2&mu;l 10x buffer (corresponding to enzymes)</li>
+
−
<li>0,5&mu;l restriction enzyme 1</li>
+
−
<li>0,5&mu;l restriction enzyme 2</li>
+
−
<li>1&mu;g DNA </li>
+
−
<li>fill up to 20&mu;l with H2O </li>
+
−
</ul>
+
 
<p>Method:</p>
 
<p>Method:</p>
−
<ul>
 
 
<li> mix all components</li>
 
<li> mix all components</li>
−
<li> incubate at 37&deg;C for 2h</li>
+
<li> incubate at 37&#176;C for 2h</li>
−
<li> heatinactivate enzymes at 80&deg;C for 10min or use for gel purification</li>
+
<li> heatinactivate enzymes at 80&#176;C for 10min or use for gel purification</li>
−
</ul></div>
+
</div>
−
 
+
</div>
−
 
+
<div class="contentRow">
−
  <label class="collapse" for="_5"><h4>> Gelpurification</h4></label>
+
<div class="contentCell contentSmallCell">
−
  <input id="_5" type="checkbox">
+
Gelpurification
−
  <div><ul>
+
</div>
 +
<div class="contentCell">
 
<p>This is a modified protocol for the Wizard SV Gel and PCR CleanUp System Kit from Promega</p>
 
<p>This is a modified protocol for the Wizard SV Gel and PCR CleanUp System Kit from Promega</p>
 
<li> Following electrophoresis, excise DNA band from gel and place gel slice in a 1,5ml microcentrifuge tube</li>
 
<li> Following electrophoresis, excise DNA band from gel and place gel slice in a 1,5ml microcentrifuge tube</li>
−
<li> Add 10&mu;l Membrane Binding Solution per 10mg of gel slice. Vortex and incubate at 5060&deg;C until gel slice is  
+
<li> Add 10&#956;l Membrane Binding Solution per 10mg of gel slice. Vortex and incubate at 5060&#176;C until gel slice is completely dissolved</li>
−
 
+
−
completely dissolved</li>
+
 
<li> Insert SV Minicolumn into Collection Tube</li>
 
<li> Insert SV Minicolumn into Collection Tube</li>
 
<li> Transfer dissolved gel mixture to the Minicolumn assembly. Incubate at room temp. for 1 minute</li>
 
<li> Transfer dissolved gel mixture to the Minicolumn assembly. Incubate at room temp. for 1 minute</li>
 
<li> Centrifuge at 16,000xg for 1 min. Discard flowthrough and reinsert Minicolumn into Collection tube</li>
 
<li> Centrifuge at 16,000xg for 1 min. Discard flowthrough and reinsert Minicolumn into Collection tube</li>
−
<li> Add 700&mu;l Membrane Wash Solution (ethanol added). Centrifuge at 16,000xg for 1 min</li>
+
<li> Add 700&#956;l Membrane Wash Solution (ethanol added). Centrifuge at 16,000xg for 1 min</li>
 
<li> Discard flowthrough and reinsert Minicolumn into Collection tube</li>
 
<li> Discard flowthrough and reinsert Minicolumn into Collection tube</li>
−
<li> Repeat this step with 500&mu;l Membrane Wash Solution. Centrifuge at 16,000xg for 5 min</li>
+
<li> Repeat this step with 500&#956;l Membrane Wash Solution. Centrifuge at 16,000xg for 5 min</li>
 
<li> Empty the Collection Tube and centrifuge the column assembly for 1,5 min</li>
 
<li> Empty the Collection Tube and centrifuge the column assembly for 1,5 min</li>
 
<li> Leave the tubes open for 10 min (to let any rest of ethanol evaporate)</li>
 
<li> Leave the tubes open for 10 min (to let any rest of ethanol evaporate)</li>
 
<li> Carefully transfer the Minicolumn to a clean 1.5ml microcentrifuge tube</li>
 
<li> Carefully transfer the Minicolumn to a clean 1.5ml microcentrifuge tube</li>
−
<li> Add 30&mu;l of NucleaseFreeWater (65&deg;C) to the Minicolumn. Incubate at room temperature for 10 min</li>
+
<li> Add 30&#956;l of NucleaseFreeWater (65&#176;C) to the Minicolumn. Incubate at room temperature for 10 min</li>
 
<li> Centrifuge at 16,000xg for 1 min.</li>
 
<li> Centrifuge at 16,000xg for 1 min.</li>
−
<li> Incubate sample at 65&deg;C for 5min</li>
+
<li> Incubate sample at 65&#176;C for 5min</li>
−
<li> Discard Minicolumn and store DNA at 4&deg;C or 20&deg;C</li>
+
<li> Discard Minicolumn and store DNA at 4&#176;C or 20&#176;C</li>
−
</ul></div>
+
</div>
−
 
+
</div>
−
 
+
<div class="contentRow">
−
  <label class="collapse" for="_6"><h4>> Ligation</h4></label>
+
<div class="contentCell contentSmallCell">
−
  <input id="_6" type="checkbox">
+
Ligation
−
  <div><ul>
+
</div>
−
<li>1&mu;l 10x ligase buffer</li>
+
<div class="contentCell">
−
<li>0,5&mu;l ligase</li>
+
<li>1&#956;l 10x ligase buffer</li>
−
<li>1&mu;l ATP</li>
+
<li>0,5&#956;l ligase</li>
−
<li>0,51&mu;l linearised vector</li>
+
<li>1&#956;l ATP</li>
−
<li>7&mu;l insert</li>
+
<li>0,51&#956;l linearised vector</li>
−
</ul>
+
<li>7&#956;l insert</li>
 
<p>Method:</p>
 
<p>Method:</p>
−
<ul>
 
 
<li>Mix all components</li>
 
<li>Mix all components</li>
−
<li>Incubate overnight at 4&deg;C</li>  
+
<li>Incubate overnight at 4&#176;C</li>  
 
<li>Gelpurify using agarose gels</li>
 
<li>Gelpurify using agarose gels</li>
−
</ul></div>
+
</div>
−
 
+
</div>
−
 
+
<div class="contentRow">
−
  <label class="collapse" for="_7"><h4>> MutagenesisPCR</h4></label>
+
<div class="contentCell contentSmallCell">
−
  <input id="_7" type="checkbox">
+
Smallscale plasmid preparation
−
  <div><ul>
+
</div>
−
<li>200ng template </li>
+
<div class="contentCell">
−
<li>1&mu;l forward primer (2mM)</li>
+
<p>Preparation was done using the PureYield™ Plasmid Miniprep System by Promega</p>
−
<li>1&mu;l reverse primer (2mM)</li>
+
−
<li>1&mu;l Phu polymerase</li>
+
−
<li>1&mu;l dNTPs</li>
+
−
<li>10&mu;l 5x Buffer</li>
+
−
<li>to 50&mu;l with H2O</li>
+
−
 
+
−
<li>Mix components</li>
+
−
<li>Run thermocycler:</li>
+
−
 
+
−
<li>98&deg;C 0:30min</li>
+
−
<li>98&deg;C 0:45min</li>
+
−
<li>55&deg;C 0:45min</li>
+
−
<li>72&deg;C 7:00 min</li>
+
−
<li>72&deg;C 1:00 min</li>
+
−
<li>4&deg;C inf </li>
+
−
</ul></div>
+
−
 
+
−
 
+
−
  <label class="collapse" for="_8"><h4>> Oligo Annealing</h4></label>
+
−
  <input id="_8" type="checkbox">
+
−
  <div><ul>
+
−
<li>2&mu;l oligo 1</li>
+
−
<li>2&mu;l oligo 2</li>
+
−
<li>96&mu;l elution buffer</li>
+
−
</ul>
+
−
<p>Method:</p>
+
−
<ul>
+
−
<li>Mix all components</li>
+
−
<li>incubate for 10 minutes at 95&deg;C</li>
+
−
<li>take tube with heat block out of heat and let it cool down to room temperature</li>
+
−
</ul></div>
+
−
 
+
−
 
+
−
  <label class="collapse" for="_9"><h4>> Smallscale plasmid preparation</h4></label>
+
−
  <input id="_9" type="checkbox">
+
−
  <div><p>Preparation was done using the PureYield™ Plasmid Miniprep System by Promega´</p>
+
−
<ul>
+
 
<li>Pick colonies for overnight cultures in 23ml LB medium with antibiotic
 
<li>Pick colonies for overnight cultures in 23ml LB medium with antibiotic
 
grow overnight</li>
 
grow overnight</li>
−
<li>Spin down 1,5ml bacteria at maximum speed for 30 minutes, discard supernatant and add further 1,5ml, spin again and  
+
<li>Spin down 1,5ml bacteria at maximum speed for 30 minutes, discard supernatant and add further 1,5ml, spin again and discard supernatant</li>
−
 
+
<li>Resuspend the bacteria pellet in 600&#956;l H2O</li>
−
discard supernatant</li>
+
<li>Add 100 &#956;l Cell Lysis Buffer, invert six times</li>
−
<li>Resuspend the bacteria pellet in 600&mu;l H2O</li>
+
<li>Add 350 &#956;l cold Neutralization Solution, mix thoroughly by inverting</li>
−
<li>Add 100 &mu;l Cell Lysis Buffer, invert six times</li>
+
−
<li>Add 350 &mu;l cold Neutralization Solution, mix thoroughly by inverting</li>
+
 
<li>Centrifuge 30s at maximum speed</li>
 
<li>Centrifuge 30s at maximum speed</li>
 
<li>Transfer supernatant to PureYield Minicolumn in collection tube</li>
 
<li>Transfer supernatant to PureYield Minicolumn in collection tube</li>
 
<li>Centrifuge for 30 sec, discard the flow-through </li>
 
<li>Centrifuge for 30 sec, discard the flow-through </li>
−
<li>Add 200 &mu;l Endotoxin Removal Wash(ERB), centrifuge for 30sec, discard the flow-through.</li>
+
<li>Add 200 &#956;l Endotoxin Removal Wash(ERB), centrifuge for 30sec, discard the flow-through.</li>
−
<li>Add 400 &mu;l Column Wash Solution(CWC), centrifuge for 30sec, discard the flow-through.</li>
+
<li>Add 400 &#956;l Column Wash Solution(CWC), centrifuge for 30sec, discard the flow-through.</li>
−
<li>Place column in eppendorf tube, add 30 &mu;l Elution Buffer to minicolumn matrix, incubate for 1 min</li>
+
<li>Place column in eppendorf tube, add 30 &#956;l Elution Buffer to minicolumn matrix, incubate for 1 min</li>
 
<li>Centrifuge column for 30s at maximum speed, flow-through contains plasmid DNA</li>
 
<li>Centrifuge column for 30s at maximum speed, flow-through contains plasmid DNA</li>
 
<li>Measure the DNA concentration using a Nanodrop</li>
 
<li>Measure the DNA concentration using a Nanodrop</li>
−
</ul></div>
+
</div>
−
 
+
</div>
−
 
+
<div class="contentRow">
−
  <label class="collapse" for="_10"><h4>> Testrestriction</h4></label>
+
<div class="contentCell contentSmallCell">
−
  <input id="_10" type="checkbox">
+
Testrestriction
−
  <div><ul>
+
</div>
−
<li>0,25&mu;l Enzyme 1</li>
+
<div class="contentCell">
−
<li>0,25&mu;l Enzyme 2</li>
+
<li>0,25&#956;l Enzyme 1</li>
−
<li>1&mu;l 10x buffer (corresponding to enzymes)</li>
+
<li>0,25&#956;l Enzyme 2</li>
 +
<li>1&#956;l 10x buffer (corresponding to enzymes)</li>
 
<li>200-300ng DNA</li>
 
<li>200-300ng DNA</li>
−
<li>Fill up with water to 10&mu;l.</li>
+
<li>Fill up with water to 10&#956;l.</li>
−
</ul>
+
 
<p>Method:</p>
 
<p>Method:</p>
−
<ul>
+
<li>Incubate at 37&#176;C for 1h</li>
−
<li>Incubate at 37&deg;C for 1h</li>
+
 
<li>Run on 1% agarose gel</li>
 
<li>Run on 1% agarose gel</li>
−
</ul>
 
 
</div>
 
</div>
−
 
+
</div>
−
<label class="collapse" for="_11"><h4>> Transformation of Bacteria</h4></label>
+
<div class="contentRow">
−
  <input id="_11" type="checkbox">
+
<div class="contentCell contentSmallCell">
−
  <div><ul>
+
Transformation of Bacteria
−
<li>50&mu;l chemically competent <i>E. coli</i></li>
+
</div>
−
<li>35&mu;l overnight ligation mix OR 1ng purified plasmid DNA</li>
+
<div class="contentCell">
−
</ul>
+
<li>50&#956;l chemically competent <i>E. coli</i></li>
 +
<li>35&#956;l overnight ligation mix OR 1ng purified plasmid DNA</li>
 
<p>Method:</p>
 
<p>Method:</p>
−
<ul>
 
 
<li> Thaw bacteria pellet on ice</li>
 
<li> Thaw bacteria pellet on ice</li>
 
<li> add ligation mixture or purified plasmid to bacteria</li>
 
<li> add ligation mixture or purified plasmid to bacteria</li>
 
<li> incubate for 20min on ice</li>
 
<li> incubate for 20min on ice</li>
−
<li> heat shock bacteria for 45s at 42&deg;C</li>
+
<li> heat shock bacteria for 45s at 42&#176;C</li>
 
<li> incubate bacteria on ice for 2min</li>
 
<li> incubate bacteria on ice for 2min</li>
−
<li> add 700&mu;l LB medium without antibiotics</li>
+
<li> add 700&#956;l LB medium without antibiotics</li>
−
<li> incubate for 1h at 37&deg;C</li>
+
<li> incubate for 1h at 37&#176;C</li>
 
<li> spin down bacteria at 2g for 2min</li>
 
<li> spin down bacteria at 2g for 2min</li>
 
<li> discard supernatant by tipping the tube</li>
 
<li> discard supernatant by tipping the tube</li>
−
<li> resuspend bacterial pellet in leftover supernatant (50100&mu;l)</li>
+
<li> resuspend bacterial pellet in leftover supernatant (50100&#956;l)</li>
 
<li> streak bacteria onto LBagar plates with antibiotics and incubate overnight </li>
 
<li> streak bacteria onto LBagar plates with antibiotics and incubate overnight </li>
−
</ul></div>
+
</div>
−
 
+
</div>
−
<label class="collapse" for="_12"><h4>> Transformation of Yeast</h4></label>
+
</div>
−
  <input id="_12" type="checkbox">
+
<div class="contentRow">
−
  <div><ul>
+
<h1>Materials</h1>
−
<li>ca. 10ml YPD</li>
+
</div>
−
<li>100&mu;l OneStep buffer</li>
+
<div class="contentRow">
−
<li>20&mu;g ssDNA</li>
+
<div class="contentCell contentSmallCell">
−
<li>100-500 ng plasmid DNA</li>
+
LB
−
<li>fresh YPD selective plate</li>
+
</div>
−
</ul>
+
<div class="contentCell">
−
<p>Method:</p>
+
<li>25g Lennox Broth</li>
−
<ul>
+
−
<li> Scrape some yeast cells off a fresh YPD plate and inoculate in liquid YPD (= culture)</li>
+
−
<li> Thaw ssDNA (e.g. salmon sperm DNA) and heat for 10 min at 95 &deg;C, then chill on ice</li>
+
−
<li> Pellet 1 mL of culture by centrifugation at > 13.000 rpm</li>
+
−
<li> Discard supernatant and resuspend cells in 100 &mu;L OneStep buffer, vortex heavily</li>
+
−
<li> Add 20 &mu;g ssDNA (10 &mu;L of 2 mg/mL) and 100  500 ng plasmid DNA to be transformed</li>
+
−
<li> Vortex and incubate at 45 &deg;C for 2 h</li>
+
−
<li> Add 1 mL YPD, mix and spin 10 sec at full speed, discard supernatant</li>
+
−
<li> Resuspend cell pellet in 1000 &mu;L YPD and plate 100 &mu;L directly on appropriate selective plates</li>
+
−
<li> Colonies appear after 2 days of incubation at 30 &deg;C</li>
+
−
</ul></div>
+
−
</div>
+
−
 
+
−
 
+
−
 
+
−
<div id="dia1" class="dia">
+
−
<br>
+
−
<label class="collapse" for="_13"><h4>> TBE </h4></label>
+
−
  <input id="_13" type="checkbox">
+
−
  <div><ul>
+
−
  <li>1M tris</li>
+
−
  <li>0,85M B(OH)3</li>
+
−
  <li>2mM EDTA</li>
+
−
  <li>pH=8,0</li>
+
−
  </ul></div>
+
−
 
+
−
<label class="collapse" for="_14"><h4>> SDS-PAGE running buffer</h4></label>
+
−
  <input id="_14" type="checkbox">
+
−
  <div><ul>
+
−
<li>20mM Tris/HCl pH 7,6</li>
+
−
<li>250mM Glycin</li>
+
−
<li>0,1% (w/v) SDS</li>
+
−
<li>in ddH2O</li>
+
−
</ul></div>
+
−
 
+
−
<label class="collapse" for="_15"><h4>> 1x Laemmli</h4></label>
+
−
  <input id="_15" type="checkbox">
+
−
  <div><ul>
+
−
<li>50mM Tris pH 6,8</li>
+
−
<li>1,25mM EDTA pH 8</li>
+
−
<li>12,5% (v/v) Glycin</li>
+
−
<li>2% (w/v) SDS</li>
+
−
<li>50mM DTT</li>
+
−
<li>2,5% (v/v) -Mercaptoethanol</li>
+
−
<li>0,025% (w/v) Bromphenolblau</li>
+
−
<li>in ddH2O</li>
+
−
</ul></div>
+
−
 
+
−
<label class="collapse" for="_16"><h4>> LB</h4></label>
+
−
  <input id="_16" type="checkbox">
+
−
  <div>
+
−
<ul>
+
−
<li>20g Lennox Broth</li>
+
 
<li>to 1l with H2O</li>
 
<li>to 1l with H2O</li>
−
</ul></div>
 
−
 
−
<label class="collapse" for="_17"><h4>> LB/Agar</h4></label>
 
−
  <input id="_17" type="checkbox">
 
−
  <div>
 
−
<ul>
 
−
<li>35g LB-Agar (Lennox)</li>
 
−
<li>to 1l H2O</li>
 
−
</ul></div>
 
−
 
−
<label class="collapse" for="_18"><h4>> SC-media</h4></label>
 
−
  <input id="_18" type="checkbox">
 
−
  <div><ul>
 
−
<li>2g yeast nitrogen base w/o amino acids</li>
 
−
<li>0,25g synthetic complete drop-out mix</li>
 
−
<li>16,5mg adenine-sulfate</li>
 
−
<li>to 300ml H2O</li>
 
−
<li>adjust pH to 5.6</li>
 
−
<li>add agar if needed </li>
 
−
</ul></div>
 
−
 
−
<label class="collapse" for="_19"><h4>> synthetic complete drop-out mix</h4></label>
 
−
  <input id="_19 type="checkbox">
 
−
  <div><ul>
 
−
<li>1g adenine hemisulfate</li>
 
−
<li>1g arginine-HCl</li>
 
−
<li>1g histidine HCl*</li>
 
−
<li>1g isoleucine</li>
 
−
<li>2g leucine*</li>
 
−
<li>2g lysine-HCl</li>
 
−
<li>2g methionine*</li>
 
−
<li>1,5g phenylalanine</li>
 
−
<li>1g serine</li>
 
−
<li>1g threonine</li>
 
−
<li>1,5g tryptophane*</li>
 
−
<li>1g tyrosine</li>
 
−
<li>0,6g uracil*</li>
 
−
<li>4,5g valine</li>
 
−
<li>for dropout mix, omit appropriate components, labelled with *</li>
 
−
<li>combine ingredients and mix thoroughly</li>
 
−
</ul>
 
 
</div>
 
</div>
−
 
−
<label class="collapse" for="_20"><h4>> YEP (yeast extract peptone)</h4></label>
 
−
  <input id="_20" type="checkbox">
 
−
  <div><ul>
 
−
<li>3g yeast extract</li>
 
−
<li>6g peptone</li>
 
−
<li>30mg adenine hemisulphate</li>
 
−
<li>300ml H2O</li>
 
−
<li>if needed, add 5g agar</li>
 
−
</ul></div>
 
−
 
−
<label class="collapse" for="_21"><h4>> 20% Glucose/Galactose/raffinose</h4></label>
 
−
  <input id="_21" type="checkbox">
 
−
  <div>
 
−
<ul>
 
−
<li>20g of appropriate sugar</li>
 
−
<li>100ml H2O</li>
 
−
</ul>
 
 
</div>
 
</div>
−
 
+
<div class="contentRow">
−
<label class="collapse" for="_22"><h4>> antibiotics concentration</h4></label>
+
<div class="contentCell contentSmallCell">
−
  <input id="_22" type="checkbox">  
+
LB-Agar
−
  <div><ul>
+
</div>
 +
<div class="contentCell">
 +
<div class="contentRow">
 +
<div class="contentCell contentSmallCell">
 +
antibiotics concentration
 +
</div>
 +
<div class="contentCell">
 
<li>chloramphenicol 34µg/ml</li>
 
<li>chloramphenicol 34µg/ml</li>
 
<li>ampicillin 100µg/ml</li>
 
<li>ampicillin 100µg/ml</li>
−
</ul></div>
+
</div>
−
 
+
</div>
−
<label class="collapse" for="_23"><h4>> One-step buffer</h4></label>
+
−
  <input id="_23" type="checkbox">
+
−
  <div><ul>
+
−
<li>0,2M LiAc</li>
+
−
<li>40% PEG4000</li>
+
−
<li>100mM DTT</li>
+
−
<li>sterile filtrated</li>
+
−
</ul></div>
+
−
 
+
−
<h2>SDS-Gels</h2>
+
−
 
+
−
 
+
−
  <label class="collapse" for="_24"><h4>> 12% separation gel</h4></label>
+
−
  <input id="_24" type="checkbox">
+
−
  <div><ul>
+
−
<li>40,5% acrylamide (30%)</li>
+
−
<li>0,375M Tris (pH 8,8)</li>
+
−
<li>1% SDS (10%)</li>
+
−
<li>1% APS(10%)</li>
+
−
<li>0,1% TEMED</li>
+
−
</ul></div>
+
−
 
+
−
  <label class="collapse" for="_25"><h4>> 5% stacking gel</h4></label>
+
−
  <input id="_25" type="checkbox">
+
−
  <div><ul>
+
−
<li>17% acrylamide (30%)</li>
+
−
<li>0,125M Tris (pH 6,8)</li>
+
−
<li>1% SDS (10%)</li>
+
−
<li>1% APS (10%)</li>
+
−
<li>0,1% TEMED</li>
+
−
</ul></div>
+
−
 
+
−
<h2>Ni-NTA buffers</h2>
+
−
<p>All buffers are from the Qiagen "Ni-NTA Agarose Purification of 6xHis-tagged Proteins from <i>E. coli</i> under Native
+
−
 
+
−
Conditions - Quick-Start"-protocol.</p>
+
−
 
+
−
  <label class="collapse" for="_26"><h4>> Lysis buffer (pH = 8)</h4></label>
+
−
  <input id="_26" type="checkbox">
+
−
  <div><ul> 
+
−
<li>50 mM NaH2PO4 </li>
+
−
<li>300 mM NaCl </li>
+
−
<li>10 mM Imidazole</li>
+
−
</ul> </div>
+
−
 
+
−
  <label class="collapse" for="_27"><h4>> Washing buffer (pH = 8)</h4></label>
+
−
  <input id="_27" type="checkbox">
+
−
  <div><ul>
+
−
<li> 50 mM NaH2PO4 </li>
+
−
<li>300 mM NaCl </li>
+
−
<li>20 mM Imidazole</li>
+
−
</ul></div>
+
−
 
+
−
  <label class="collapse" for="_28"><h4>> Elution buffer (pH = 8)</h4></label>
+
−
  <input id="_28" type="checkbox">
+
−
  <div><ul>
+
−
<li>50 mM NaH2PO4</li>
+
−
<li>300 mM NaCl</li>
+
−
<li>250 mM Imidazole</li>
+
−
</ul></div>
+
−
</div>
+
−
 
+
−
 
+
−
<div id="dia2" class="dia">
+
−
<br>
+
−
<h2>Experiments</h2>
+
−
 
+
−
 
+
−
 
+
−
<label class="collapse" for="_29"><h4>> Agarose Gels</h4></label>
+
−
 
+
−
  <input id="_29" type="checkbox">
+
−
 
+
−
  <div><ul>
+
−
 
+
−
 
+
−
 
+
−
<li> cast gels</li>
+
−
 
+
−
<li> load samples with loading dye</li>
+
−
 
+
−
<li> run gels at 120 V</li></ul></div>
+
−
 
+
−
 
+
−
 
+
−
<label class="collapse" for="_30"><h4>> Expression</h4></label>
+
−
 
+
−
  <input id="_30" type="checkbox">
+
−
 
+
−
  <div><ul>
+
−
 
+
−
 
+
−
<li> pETue-NAGA-Intein was transformed into <i>E. coli<i> BL21(DE3)</li>
+
−
 
+
−
<li> grow culture to OD_600 = 0.3</li>
+
−
 
+
−
<li> induce with 0.2 mM IPTG</li>
+
−
 
+
−
<li> express O/N at room temperature</li>
+
−
 
+
−
<li> centrifuge at 6000 g for 30 minutes at 4°C</li>
+
−
 
+
−
<li> resuspend pellet in 10 ml lysis buffer</li>
+
−
 
+
−
<li> sonicate for a total of 3 minutes (40% amplitude, 1 s intervals)</li></ul></div>
+
−
 
+
−
 
+
−
<label class="collapse" for="_31"><h4>> Fluorescence assay</h4></label>
+
−
 
+
−
  <input id="_31" type="checkbox">
+
−
 
+
−
  <div><ul>
+
−
 
+
−
 
+
−
 
+
−
<li>Cells were measured undiluted or in 1:2 dilution in 96 well plates (black, flat bottom)</li>
+
−
 
+
−
<li>Following excitation and emission wavelengths were used for the different fluorescent proteins:</li>
+
−
 
+
−
<li>GFP <ul>
+
−
 
+
−
<li>Exc: 488 nm</li>
+
−
 
+
−
<li>Em: 520 nm</li></ul>
+
−
 
+
−
<li>Dronpa</li><ul>
+
−
 
+
−
<li>Exc: 503 nm</li>
+
−
 
+
−
<li>Em: 535 nm</li></ul>
+
−
 
+
−
<li>RFP<ul>
+
−
 
+
−
<li>Exc: 584 nm</li>
+
−
 
+
−
<li>Em: 616 nm</li></ul>
+
−
 
+
−
<li>Fluorescence was measured with Tecan plate reader (Infinite M200) and analysed with Tecan i-control v1.10</ul></div>
+
−
 
+
−
 
+
−
<label class="collapse" for="_32"><h4>> Fluorescence Microscopy</h4></label>
+
−
 
+
−
  <input id="_32" type="checkbox">
+
−
 
+
−
  <div><ul>
+
−
 
+
−
 
+
−
<h4> Sample preparation </h4>
+
−
 
+
−
 
+
−
 
+
−
<li>A thin layer of low melting agarose was supplied on glass slides </li>
+
−
 
+
−
<li>10 µl Yeast cells grown overnight were inoculated in the solidified agarose </li>
+
−
 
+
−
<li>samples were used for microscopy after at least 30min incubation at 30° in a humid environment </li>
+
−
 
+
−
 
+
−
<h4> Microscopy Setup </h4>
+
−
 
+
−
 
+
−
<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>
+
−
 
+
−
 
+
−
<h4> Dronpa Illumination </h4>
+
−
 
+
−
 
+
−
<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>
+
−
 
+
−
<label class="collapse" for="_33"><h4>> Luciferase Assay</h4></label>
+
−
 
+
−
  <input id="_33" type="checkbox">
+
−
 
+
−
  <div><ul>
+
−
 
+
−
 
+
−
 
+
−
<li>Grow overnight culture of yeast: Induction or repression of luciferase expression by addition of different
+
−
 
+
−
galactose/glucose/raffinose concentrations for pGal or pSUC promoters.</li>
+
−
 
+
−
<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>
+
−
 
+
−
<li>Firefly luciferase protocol: mix either cell suspension or cell lysate 1:1 with reconstituted luciferase assay
+
−
 
+
−
reagent</li>
+
−
 
+
−
<li>Luciferase activity measured with Tecan plate reader (Infinite M200) and analysed with Tecan i-control v1.10.
+
−
 
+
−
</li></ul></div>
+
−
 
+
−
<label class="collapse" for="_36"><h4>> Fluorescent-peptide-coupling Assay</h4></label>
+
−
 
+
−
  <input id="_36" type="checkbox">
+
−
 
+
−
  <div><ul>
+
−
 
+
−
 
+
−
<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>
+
−
 
+
−
<li> dilute with 110 µl of 2x Laemmli buffer</li>
+
−
 
+
−
<li> incubate at 95°C for 10 minutes</li>
+
−
 
+
−
<li> load onto SDS polyacrylamide gel</li></ul></div>
+
−
 
+
−
<label class="collapse" for="_34"><h4>> MALDI-TOF mass spectrometry</h4></label>
+
−
 
+
−
  <input id="_34" type="checkbox">
+
−
 
+
−
  <div><ul>
+
−
 
+
−
 
+
−
Preparation and trypsin digest:
+
−
 
+
−
<li> cut bands from SDS polyacrylamide gel</li>
+
−
 
+
−
<li> cover gel piece with Buffer2 for 30 min to discolor</li>
+
−
 
+
−
<li> cover with acetonitrile (ACN) for 10 min to dehydrate</li>
+
−
 
+
−
<li> cover with Buffer1 containing 10 mM DTT to reduce for 45 min</li>
+
−
 
+
−
<li> cover with Buffer1 containing 55 mM iodoacetamide (IAA) to alkylate for 30 min</li>
+
−
 
+
−
<li> cover with Buffer1 for 15 min to wash</li>
+
−
 
+
−
<li> cover with Buffer2 for 10 min to wash</li>
+
−
 
+
−
<li> cover with 100% ACN for 10 min to dehydrate</li>
+
−
 
+
−
<li> cover with 20 µl Buffer1 containing 100 ng Trypsin</li>
+
−
 
+
−
<li> digest for 5 h at 37 °C</li>
+
−
 
+
−
<li> stop reaction by addition of 2 µl of 10% TFA</li>
+
−
 
+
−
<li> extract peptides by incubation for 30 minutes</li>
+
−
 
+
−
 
+
−
Buffer1: 50 mM ammoniumbicarbonat (AB)
+
−
Buffer2: 70% 50 mM AB, 30% ACN
+
−
 
+
−
 
+
−
Measurement:
+
−
 
+
−
<li> mix 1  ml of sample with 1 µl of gentisic acid matrix solution</li>
+
−
 
+
−
<li> pipette unto target</li>
+
−
 
+
−
<li> wait for crystallisation</li>
+
−
 
+
−
<li> measure with MALDI-TOF</li>
+
−
 
+
−
<li> analyse data using FlexAnalysis und FlexControl</li>
+
−
 
+
−
 
+
−
The result score is calculated as Score = -log10(P).</ul></div>
+
−
 
+
−
 
+
−
 
+
−
 
+
−
<label class="collapse" for="_34"><h4>> Ni-NTA Purification</h4></label>
+
−
 
+
−
  <input id="_34" type="checkbox">
+
−
 
+
−
  <div><ul>
+
−
 
+
−
 
+
−
Pellet
+
−
 
+
−
<li> centrifugation of the bacterial culture for 10 min at 8000 rpm at 4 °C</li>
+
−
 
+
−
<li> discard the supernatant</li>
+
−
 
+
−
Lysis
+
−
 
+
−
<li> per 1g wet weight of the pellet add 4ml lysis buffer</li>
+
−
 
+
−
<li> fill up to 0.5 mg/ml with lysozyme</li>
+
−
 
+
−
<li> incubation on ice for 2 h</li>
+
−
 
+
−
<li> centrifuge for 30 min at 10000 g at 4 °C</li>
+
−
 
+
−
Column
+
−
 
+
−
<li> equilibrate the column with 0.5 ml Ni-NTA (0.25 ml bed volume)</li>
+
−
 
+
−
<li> add 0.5 ml lysis buffer and slightly centrifuge the buffer through the column, it has to stay wet (do twice)</li>
+
−
 
+
−
<li> pump the cell lysate (supernatant) for 1 h at ice through the column</li>
+
−
 
+
−
Wash
+
−
 
+
−
<li> twice with 1.25ml wash buffer</li>
+
−
 
+
−
<li> four times with 0.5 ml elution buffer</li>
+
−
 
+
−
<li> recover the supernatant</li>
+
−
 
+
−
 
+
−
 
+
−
<li> Load a sample of every step in an SDS gel.</li></ul></div>
+
−
 
+
−
 
+
−
 
+
−
 
+
−
 
+
−
 
+
−
<label class="collapse" for="_35"><h4>> Purification</h4></label>
+
−
 
+
−
  <input id="_35" type="checkbox">
+
−
 
+
−
  <div><ul>
+
−
 
+
−
 
+
−
<li> centrifuge lysate at 15,000 g for 30 min</li>
+
−
 
+
−
<li> purify supernatant with Ni-NTA beads (native conditions, gravity flow)</li>
+
−
 
+
−
<li> elute once with 500 µl elution buffer</li></ul></div>
+
−
 
+
−
 
+
−
<label class="collapse" for="_27"><h4>> SDS-page</h4></label>
+
−
 
+
−
  <input id="_27" type="checkbox">
+
−
 
+
−
  <div><ul>
+
−
 
+
−
 
+
−
 
+
−
<li>Cast gels with composition as described <here> (Link bitte einfügen zu der Zusammensetzung)</li>
+
−
 
+
−
<li>Load samples with SDS-PAGE loading dye </li>
+
−
 
+
−
<li>Run gels at 150V until front is run through the gel</li></ul></div>
+
−
 
+
−
 
+
−
 
+
−
+
 
</div>
 
</div>
 
</html>
 
</html>

Revision as of 15:54, 19 October 2016

Methods

Chemically Competent E. coli Cells
  • autoclave 250mL LB in Erlenmeyer beaker
  • plate cells from cell stock on agarose plate with appropriate antibiotics
  • inoculate one clone in 5mL of LB with antibiotics, grow at 37°C o/n
  • expand to 500mL and grow until OD600nm = 0.35
  • transfer into 50mL Falcon tubes
  • refrigerate centrifuge at 4°C, chill 2x 25mL 0.1M CaCl2 (Falcon tubes) and 5mL 0.1M CaCl2 with 10% Glycerol
  • spin down cells at 2000g for 10Min at 4°C
  • resuspend 5 Falcon cell pellets in 25mL ice cold 0.1M CaCl2
  • spin down at 2000g for 10Min at 4°C
  • resuspend pellet in 2x5mL (=10mL) ice cold 0.1M CaCl2 with 10% Glycerol
  • freeze 50-100μl aliquots
  • store at 20°C
    • DoubleDigest Restriction of gBlocks and BioBricks
  • 2μl 10x buffer (corresponding to enzymes)
  • 0,5μl restriction enzyme 1
  • 0,5μl restriction enzyme 2
  • 1μg DNA
  • fill up to 20μl with H2O

    • Method:

  • mix all components
  • incubate at 37°C for 2h
  • heatinactivate enzymes at 80°C for 10min or use for gel purification
    • Gelpurification

    This is a modified protocol for the Wizard SV Gel and PCR CleanUp System Kit from Promega

  • Following electrophoresis, excise DNA band from gel and place gel slice in a 1,5ml microcentrifuge tube
  • Add 10μl Membrane Binding Solution per 10mg of gel slice. Vortex and incubate at 5060°C until gel slice is completely dissolved
  • Insert SV Minicolumn into Collection Tube
  • Transfer dissolved gel mixture to the Minicolumn assembly. Incubate at room temp. for 1 minute
  • Centrifuge at 16,000xg for 1 min. Discard flowthrough and reinsert Minicolumn into Collection tube
  • Add 700μl Membrane Wash Solution (ethanol added). Centrifuge at 16,000xg for 1 min
  • Discard flowthrough and reinsert Minicolumn into Collection tube
  • Repeat this step with 500μl Membrane Wash Solution. Centrifuge at 16,000xg for 5 min
  • Empty the Collection Tube and centrifuge the column assembly for 1,5 min
  • Leave the tubes open for 10 min (to let any rest of ethanol evaporate)
  • Carefully transfer the Minicolumn to a clean 1.5ml microcentrifuge tube
  • Add 30μl of NucleaseFreeWater (65°C) to the Minicolumn. Incubate at room temperature for 10 min
  • Centrifuge at 16,000xg for 1 min.
  • Incubate sample at 65°C for 5min
  • Discard Minicolumn and store DNA at 4°C or 20°C
    • Ligation
  • 1μl 10x ligase buffer
  • 0,5μl ligase
  • 1μl ATP
  • 0,51μl linearised vector
  • 7μl insert

    • Method:

  • Mix all components
  • Incubate overnight at 4°C
  • Gelpurify using agarose gels
    • Smallscale plasmid preparation

    Preparation was done using the PureYield™ Plasmid Miniprep System by Promega

  • Pick colonies for overnight cultures in 23ml LB medium with antibiotic grow overnight
  • Spin down 1,5ml bacteria at maximum speed for 30 minutes, discard supernatant and add further 1,5ml, spin again and discard supernatant
  • Resuspend the bacteria pellet in 600μl H2O
  • Add 100 μl Cell Lysis Buffer, invert six times
  • Add 350 μl cold Neutralization Solution, mix thoroughly by inverting
  • Centrifuge 30s at maximum speed
  • Transfer supernatant to PureYield Minicolumn in collection tube
  • Centrifuge for 30 sec, discard the flow-through
  • Add 200 μl Endotoxin Removal Wash(ERB), centrifuge for 30sec, discard the flow-through.
  • Add 400 μl Column Wash Solution(CWC), centrifuge for 30sec, discard the flow-through.
  • Place column in eppendorf tube, add 30 μl Elution Buffer to minicolumn matrix, incubate for 1 min
  • Centrifuge column for 30s at maximum speed, flow-through contains plasmid DNA
  • Measure the DNA concentration using a Nanodrop
    • Testrestriction
  • 0,25μl Enzyme 1
  • 0,25μl Enzyme 2
  • 1μl 10x buffer (corresponding to enzymes)
  • 200-300ng DNA
  • Fill up with water to 10μl.

    • Method:

  • Incubate at 37°C for 1h
  • Run on 1% agarose gel
    • Transformation of Bacteria
  • 50μl chemically competent E. coli
  • 35μl overnight ligation mix OR 1ng purified plasmid DNA

    • Method:

  • Thaw bacteria pellet on ice
  • add ligation mixture or purified plasmid to bacteria
  • incubate for 20min on ice
  • heat shock bacteria for 45s at 42°C
  • incubate bacteria on ice for 2min
  • add 700μl LB medium without antibiotics
  • incubate for 1h at 37°C
  • spin down bacteria at 2g for 2min
  • discard supernatant by tipping the tube
  • resuspend bacterial pellet in leftover supernatant (50100μl)
  • streak bacteria onto LBagar plates with antibiotics and incubate overnight

    • Materials

    LB
  • 25g Lennox Broth
  • to 1l with H2O
    • LB-Agar
    antibiotics concentration
  • chloramphenicol 34µg/ml
  • ampicillin 100µg/ml