Difference between revisions of "Team:Warwick/Cloning"

 
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<br>5. <a href="http://synbio.tsl.ac.uk/golden-gate-assembly-protocol/">Golden gate assembly</a> of plasmid backbones   
 
<br>5. <a href="http://synbio.tsl.ac.uk/golden-gate-assembly-protocol/">Golden gate assembly</a> of plasmid backbones   
 
and full construct.
 
and full construct.
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<br>6. Transform electrocompetent E. <i>coli</i> cells with full plasmid
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<br>6. Transform electrocompetent <i>E.coli</i> cells with full plasmid
 
<br>7. Grow cells
 
<br>7. Grow cells
 
<br>8. Miniprep full construct plasmid from modified cells
 
<br>8. Miniprep full construct plasmid from modified cells
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<br>10. PCR the rest of the anchor proteins from the G-blocks ordered (INP, PGSA, and BCLA)
 
<br>10. PCR the rest of the anchor proteins from the G-blocks ordered (INP, PGSA, and BCLA)
 
<br>11. Ligate the anchor proteins into digested full construct plasmid
 
<br>11. Ligate the anchor proteins into digested full construct plasmid
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<br>12. Transform all plasmids into electrocompetent E. <i>coli</i> - end product: four different types of cell, each expressing the full construct with slightly different transmembrane domains
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<br>12. Transform all plasmids into electrocompetent <i>E.coli</i> - end product: four different types of cell, each expressing the full construct with slightly different transmembrane domains
 
<br>13. Prepare transformed cells for microscopy (mount cells on glass slides)  
 
<br>13. Prepare transformed cells for microscopy (mount cells on glass slides)  
 
<br>14. Bind antibodies to FLAG tag - measure under fluorescent microscope
 
<br>14. Bind antibodies to FLAG tag - measure under fluorescent microscope
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<br>15. Determine whether the gene is being expressed and whether the expressed protein is being transported and is binding to the membrane of the E. <i>coli</i>
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<br>15. Determine whether the gene is being expressed and whether the expressed protein is being transported and is binding to the membrane of the <i>E.coli</i>
 
<br>16. Compare intensities - select anchor protein that fluoresces brightest, this anchor protein with be used for all subsequent experiments.
 
<br>16. Compare intensities - select anchor protein that fluoresces brightest, this anchor protein with be used for all subsequent experiments.
 
</p>
 
</p>
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<br>3. PCR zinc finger genes from the G-blocks ordered
 
<br>3. PCR zinc finger genes from the G-blocks ordered
 
<br>4. Golden gate assembly of digested full construct plasmid and the three zinc finger PCR products
 
<br>4. Golden gate assembly of digested full construct plasmid and the three zinc finger PCR products
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<br>5. Transform electrocompetent E. <i>coli</i> with each modified plasmid - end product: four different types of cells, each with the same anchor protein but different zinc finger DNA binding domains
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<br>5. Transform electrocompetent <i>E.coli</i> with each modified plasmid - end product: four different types of cells, each with the same anchor protein but different zinc finger DNA binding domains
 
<br>6. Prepare the cells for microscopy by binding them to slides
 
<br>6. Prepare the cells for microscopy by binding them to slides
 
<br>7. Prepare four different sets of fluorescent oligos that each contain the target sequence for a zinc finger
 
<br>7. Prepare four different sets of fluorescent oligos that each contain the target sequence for a zinc finger
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<p>
 
<p>
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<img src="https://static.igem.org/mediawiki/2015/4/45/Warwick_Diagram_of_experiment_3.jpeg" align="middle">
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<img src="https://static.igem.org/mediawiki/2015/4/45/Warwick_Diagram_of_experiment_3.jpeg" align="middle">
 
</p>
 
</p>
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</div>
 
</div>

Latest revision as of 17:51, 17 September 2015

Warwick iGEM 2015

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