Difference between revisions of "Team:AshesiGhana/Protocols"

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<p>
 
<p>
<b>Materials </b><br>
+
<b>The iGEM Registry Transformation Protocol</b><br>
Resuspended DNA<br>
+
<a>Please click on the link to view the  <a href="https://static.igem.org/mediawiki/2017/8/8e/T--Northwestern--iGEMTransformation.pdf">Protocol for Transformations</a><br>
10pg/ul Control DNA<br>
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Competent Cells<br>
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2ml Microtubes<br>
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Floating Foam Tubes<br>
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Ice $ Ice Bucket<br>
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Lab Timer 42 °C water bath<br>
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SOC Media<br>
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37C Incubator<br>
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Pertri plates w/ LB agar and antibiotic<br>
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Sterile Spreader<br>
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Pipettes and Tips<br>
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<br>
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<b>Preparation of chemical competent cells:</b>
+
<ol style="font-size:16px;">
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<li>Thaw competent cells on ice<br></li>
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<li>Pipette 50 micto liters of competent cells into 2ml tube<br></li>
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<li>Pipette 1 micro litre of resuspended DNA into 2ml tube<br></li>
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<li>Pipette 1 micro litre of control DNA into 2ml tube<br></li>
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<li>Close 2ml tubes, incubate on ice for 30min<br></li>
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<li>Heat shock tubes at 42C for 1 min<br></li>
+
<li>Incubate on ice for 5min<br></li>
+
</ol>
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</p>
+
  
 
<p>
 
<b>Chemical Transformation:</b>
 
<ol style="font-size:16px;">
 
 
<li>Pipette 200microlitre SOC media to each transformation <br></li>
 
<li>Incubate at 37C for 2 hours, shaker or rotor recommended<br></li>
 
<li>Pipette each transformation on 2 petri plates for a 20 micro litre and 200 micro litre plating <br></li>
 
<li>Incubate transformations overnight<br></li>
 
<li>APick single colonies<br></li>
 
<li>Count colonies for control transformation<br></li>
 
</ol>
 
 
</p>
 
</p>
 
  
 
     </div>
 
     </div>
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<p>
 
<p>
<b>Materials (consumables):</b><br>
+
<b>The Plasmid Digest Protocol</b><br>
5µl NEB buffer 2 <br>
+
<a>Please click on the link to view the  <a href="https://static.igem.org/mediawiki/2017/3/32/T--AshesiGhana--plasmid.pdf">Protocol for the Plasmid Digest</a><br>
0.5µl EcoRI-HF <br>
+
 
0.5µl PstI<br>
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19µl dH20 <br>
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Ice<br>
+
<br>
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<b>Methods:</b><br>
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<ol style="font-size:16px;">
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<li>Add 4µl linearized plasmid backbone (25ng/µl for 100ng total) </li>
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<li>Add 4µl of Enzyme Master Mix </li>
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<li>MDigest 37⁰C/30min, heat kill 80⁰C/20min.</li>
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</ol>
+
 
</p>
 
</p>
 
 
  
 
   </div>
 
   </div>
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<div>
 
<div>
                     <div class="col-md-11">Part Digest</div><div class="col-md-1"><i class="fa" aria-hidden="true"></i></div>
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                     <div class="col-md-11">Interlab Plate Reader</div><div class="col-md-1"><i class="fa" aria-hidden="true"></i></div>
 
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<p>
 
<p>
<b>Materials (consumables):</b><br>
+
<b>The Interlab Plate Reader Protocol</b><br>
5µl NEB Buffer 2 <br>
+
<a>Please click on the link to view the  <a href="https://static.igem.org/mediawiki/2017/4/43/T--AshesiGhana--interlab.pdf">Protocol</a><br>
0.5µl EcoRI-HF<br>
+
 
0.5µL SpeI <br>
+
</p>
DNA template<br>
+
19µl dH2O <br>
+
<br>
+
<b>Methods:</b><br>
+
<ol style="font-size:16px;">
+
<li>Add 10µl of part DNA (100ng total) </li>
+
<li>Add 4µl of enzyme Mix </li>
+
<li>Digest 37⁰C/30min, heat kill 80⁰C/20min </li>
+
  
 
   </div>
 
   </div>
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<p>
 
<p>
<b>Materials (consumables):</b><br>
+
<b>The Preparation Protocol for LB Agar and LB Chloramphemicol Plates</b><br>
LB broth <br>
+
<a>Please click on the link to view the  <a href="https://static.igem.org/mediawiki/2017/3/3f/T--AshesiGhana--lbagar.pdf">Protocol</a><br>
Bacteriological Agar <br>
+
100mg/ml Chloramphenicol Solution <br>
+
  
<br>
 
<b>PREPARATION OF LB-AGAR PLATE (500ml) </b><br>
 
<ol style="font-size:16px;">
 
<li>Add 12.5g of LB broth powder into an appropriate bottle</li>
 
<li>Add 7.5g of Agar </li>
 
<li>Autoclave </li>
 
<li>Pour hot/ warm (melted) media at 20ml per petri dish</li>
 
</ol>
 
 
<br>
 
<b>PREPARATION OF LB-AMP PLATE (500ml) </b><br>
 
<ol style="font-size:16px;">
 
<li>Add 12.5g of LB broth powder into an appropriate bottle  2</li>
 
<li>Add 7.5g of Agar </li>
 
<li>Autoclave </li>
 
<li> Allow media to cool till it can be felt at the back of the palm without burning (but still not solidified). </li>
 
 
<li> Add 500µl of 100mg/ml Chloramphenicol solution  </li>
 
 
<li>  Swirl to mix the antibiotic uniformly in the media </li>
 
<li>  Pour the melted media at 20ml per petri dish</li>
 
</ol>
 
 
</p>
 
</p>
  
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<p>
 
<p>
<b>Materials (consumables):</b><br>
+
<b>The Zyppy™ Plasmid Miniprep Protocol</b><br>
7X Lysis Buffer*1 (Blue) <br>
+
<a>Please click on the link to view the <a href="https://static.igem.org/mediawiki/2017/6/63/T--AshesiGhana--zyppy.pdf">Protocol for the Plasmid Digest</a><br>
Neutralization Buffer*2 (Yellow) <br>
+
Endo-Wash Buffer <br>
+
Zyppy™ Elution Buffer <br>
+
Collection Tubes<br>
+
<br>
+
<b>Methods:</b><br>
+
<ol style="font-size:16px;">
+
<li>Add 600 µl of bacterial culture grown in LB medium to a 1.5 ml microcentrifuge tube. </li>
+
<li>Add 100 µl of 7X Lysis Buffer (Blue)1 and mix by inverting the tube 4-6 times. Proceed to step 3 within 2 minutes. </li>
+
<li>Add 350 µl of cold Neutralization Buffer (Yellow) and mix thoroughly</li>
+
<li>Place the column into a Collection Tube and centrifuge for 15 seconds. </li>
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<li>Discard the flow-through and place the column back into the same Collection Tube. </li>
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<li>Add 200 µl of Endo-Wash Buffer to the column</li>
+
<li>Add 400 µl of Zyppy™ Wash Buffer to the column</li>
+
<li> Transfer the column into a clean 1.5 ml microcentrifuge tube then add 30 µl of Zyppy™ Elution Buffer2 directly to the column matrix and let stand for one minute at room temperature.
+
</li>
+
<li>. Centrifuge for 30 seconds to elute the plasmid DNA. </li>
+
 
+
</ol>
+
  
 +
</p>
 
   </div>
 
   </div>
 
</div>
 
</div>

Revision as of 09:33, 1 November 2017

This page shows general protocols given by the suppliers, supervisors and/or published papers.