Difference between revisions of "Team:AshesiGhana/Protocols"

 
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<p>
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<b>Materials </b><br>
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<b>The iGEM Registry Transformation Protocol</b><br>
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Resuspended DNA<br>
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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>
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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>
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<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>
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<li>Incubate on ice for 5min<br></li>
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</ol>
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</p>
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<p>
 
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<b>Chemical Transformation:</b>
 
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<ol style="font-size:16px;">
 
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<li>Pipette 200microlitre SOC media to each transformation <br></li>
 
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<li>Incubate at 37C for 2 hours, shaker or rotor recommended<br></li>
 
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<li>Pipette each transformation on 2 petri plates for a 20 micro litre and 200 micro litre plating <br></li>
 
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<li>Incubate transformations overnight<br></li>
 
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<li>APick single colonies<br></li>
 
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<li>Count colonies for control transformation<br></li>
 
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</ol>
 
 
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<p>
 
<p>
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<b>Materials (consumables):</b><br>
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<b>The Plasmid Digest Protocol</b><br>
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5µl NEB buffer 2 <br>
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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>
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0.5µl EcoRI-HF <br>
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0.5µl PstI<br>
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19µl dH20 <br>
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Ice<br>
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<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>
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                     <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>
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<b>Materials (consumables):</b><br>
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<b>The Interlab Plate Reader Protocol</b><br>
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5µl NEB Buffer 2 <br>
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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>
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0.5µl EcoRI-HF<br>
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0.5µL SpeI <br>
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</p>
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DNA template<br>
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19µl dH2O <br>
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<br>
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<b>Methods:</b><br>
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<ol style="font-size:16px;">
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<li>Add 10µl of part DNA (100ng total) </li>
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<li>Add 4µl of enzyme Mix </li>
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<li>Digest 37⁰C/30min, heat kill 80⁰C/20min </li>
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<p>
 
<p>
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<b>Materials (consumables):</b><br>
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The Preparation Protocol for LB Agar and LB Chloramphemicol Plates</b><br>
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LB broth <br>
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<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>
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Bacteriological Agar <br>
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100mg/ml Chloramphenicol Solution <br>
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<br>
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</p>
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<b>PREPARATION OF LB-AGAR PLATE (500ml) </b><br>
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<ol style="font-size:16px;">
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<li>Add 12.5g of LB broth powder into an appropriate bottle</li>
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<li>Add 7.5g of Agar </li>
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<li>Autoclave </li>
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<li>Pour hot/ warm (melted) media at 20ml per petri dish</li>
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</ol>
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<br>
 
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<b>PREPARATION OF LB-AMP PLATE (500ml) </b><br>
 
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<ol style="font-size:16px;">
 
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<li>Add 12.5g of LB broth powder into an appropriate bottle  2</li>
 
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<li>Add 7.5g of Agar </li>
 
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<li>Autoclave </li>
 
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<li> Allow media to cool till it can be felt at the back of the palm without burning (but still not solidified). </li>
 
  
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<li> Add 500µl of 100mg/ml Chloramphenicol solution  </li>
 
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<li>  Swirl to mix the antibiotic uniformly in the media </li>
 
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<li>  Pour the melted media at 20ml per petri dish</li>
 
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</ol>
 
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</p>
 
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<p>
 
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<b>Run gel:</b>
 
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<ol style="font-size:16px;">
 
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<li>Add 5µl of PCR solution and 1µl 10x loading dye</li>
 
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<li>Load 6µl of DNA ladder alongside and all samples (<b>Do not forget to add dye to ladder too</b>) - NEB 1kb ladder used</li>
 
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<li>Run gel at 100V for 45 min</li>
 
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<li>Visualise gel on a transilluminator (SYBR Safe binds DNA and fluoresces under UV light)
 
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</ol>
 
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<p>
 
<p>
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<b>Materials (consumables):</b><br>
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<b>The Zyppy™ Plasmid Miniprep Protocol</b><br>
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7X Lysis Buffer*1 (Blue) <br>
+
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>
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Neutralization Buffer*2 (Yellow) <br>
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Endo-Wash Buffer <br>
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Zyppy™ Elution Buffer <br>
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Collection Tubes<br>
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<br>
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<b>Methods:</b><br>
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<ol style="font-size:16px;">
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<li>Add 600 µl of bacterial culture grown in LB medium to a 1.5 ml microcentrifuge tube. </li>
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<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>
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<li>Add 350 µl of cold Neutralization Buffer (Yellow) and mix thoroughly</li>
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<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>
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<li>Add 400 µl of Zyppy™ Wash Buffer to the column</li>
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<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.
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</li>
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<li>. Centrifuge for 30 seconds to elute the plasmid DNA. </li>
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</ol>
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Latest revision as of 09:37, 1 November 2017

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

The iGEM Registry Transformation Protocol
Please click on the link to view the Protocol for Transformations

The Plasmid Digest Protocol
Please click on the link to view the Protocol for the Plasmid Digest

The Interlab Plate Reader Protocol
Please click on the link to view the Protocol

The Preparation Protocol for LB Agar and LB Chloramphemicol Plates
Please click on the link to view the Protocol

The Zyppy™ Plasmid Miniprep Protocol
Please click on the link to view the Protocol for the Plasmid Digest