Difference between revisions of "Team:ASIJ Tokyo/Experiments"

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             <ul>
 
             <ul>
 
                 <li> <b>&emsp;&emsp;&emsp; Goal:</b>
 
                 <li> <b>&emsp;&emsp;&emsp; Goal:</b>
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&emsp;&emsp;&emsp; The sialic acid assay was performed to determine the sialic acid levels secreted by each construct. Based on previous literature, we determined that the unmutated constructs would secrete more sialic acid. On the other hand, the mutated constructs were expected to secrete less, due to its tendency to polymerize in the cell, preventing its ability to exit the cell. This assay would allow us to determine the sialic acid levels for each construct, and thus allow us to compare the CRISPR edited construct with unmutated construct, to determine if our gene edit was successful. If successful, the CRISPR edited constructs would show sialic acid secretion similar to that of the unmutated construct. </li>
+
<br>The sialic acid assay was performed to determine the sialic acid levels secreted by each construct. Based on previous literature, we determined that the unmutated constructs would secrete more sialic acid. On the other hand, the mutated constructs were expected to secrete less, due to its tendency to polymerize in the cell, preventing its ability to exit the cell. This assay would allow us to determine the sialic acid levels for each construct, and thus allow us to compare the CRISPR edited construct with unmutated construct, to determine if our gene edit was successful. If successful, the CRISPR edited constructs would show sialic acid secretion similar to that of the unmutated construct. </li>
 
                 <li> <b>&emsp;&emsp;&emsp; Why:</b>
 
                 <li> <b>&emsp;&emsp;&emsp; Why:</b>
−
&emsp;&emsp;&emsp; We decided after finishing the behaviour control lab that we should seek quantitative results that will helps us more accurately determine if the gene edit was successful. We had theories several methods to obtain quantitative results but in the end decided on the Sialic assay because we already had the materials to perform it. </li>
+
<br> We decided after finishing the behaviour control lab that we should seek quantitative results that will helps us more accurately determine if the gene edit was successful. We had theories several methods to obtain quantitative results but in the end decided on the Sialic assay because we already had the materials to perform it. </li>
 
             </ul>
 
             </ul>
 
     </ul>
 
     </ul>

Revision as of 04:57, 16 October 2018

EXPERIMENTS

Part I CRISPR


Part II CRISPR Confirmation (genotypic)


Part III: Designing of the new constructs (this is just modeling the construct design and not really a protocol)


Part IV: Gibson Assembly of Constructs


Part V: Deciding which fluorescent marker to use (water melon tubes, and plates)..deciding then to only use the GFP ones


Part VI: Genotypic and Phenotypic Proof of Concept

  • a. Agarose gel electrophoresis of plasmid constructs (agarose gel electorphoresis protocol)
  • b. Phenotypic Proof (Transformation and growing on selection plates)

Part VII: Quantifying Protein

  • a. GFP protein purification using column chromatography
  • b. Sialic Acid Assay - original version of protocol
    •     Goal:
      The sialic acid assay was performed to determine the sialic acid levels secreted by each construct. Based on previous literature, we determined that the unmutated constructs would secrete more sialic acid. On the other hand, the mutated constructs were expected to secrete less, due to its tendency to polymerize in the cell, preventing its ability to exit the cell. This assay would allow us to determine the sialic acid levels for each construct, and thus allow us to compare the CRISPR edited construct with unmutated construct, to determine if our gene edit was successful. If successful, the CRISPR edited constructs would show sialic acid secretion similar to that of the unmutated construct.
    •     Why:
      We decided after finishing the behaviour control lab that we should seek quantitative results that will helps us more accurately determine if the gene edit was successful. We had theories several methods to obtain quantitative results but in the end decided on the Sialic assay because we already had the materials to perform it.

Part VIII: Incorporation of New information to use His-tag