Team:Cambridge-JIC/Constructs/gal4 hap1gr H2RBmRFP

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Constructs:
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<div align="center"><a href="https://2014.igem.org/wiki/index.php?title=Team:Cambridge-JIC/Constructs/gal4_hap1gr_H2RBmRFP&action=edit">Edit this page.</a></div>
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<h1> Gal4 - Hap1GR - H2BmRFP</h1>
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<h2> Design </h2>
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<div id="Aim" align="left">
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<h4> Aim of the construct </h4>
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<ul>
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<li> We'll try out 35s->GAL4->HAP1GR->H2BmRFP. </li>
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<li> Since H2BmRFP has not been tried in Mp, we'll also try 35s->H2BmRFP. A positive control for lab technique will be 35s->Venus N7</li>
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<li> We'll also use FF's original plasmid to try 35s-Hap1GR-mRFP, as a control for the Hap1GR. I expect low transformation rate as in the original plasmid, we're using 35s as promoter for HygR selection marker.
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<li> Not sure whether 35s->Gal4 has been tried (check with BP), so 35s-Gal4-venus n7 is doable with BP's construct as a control </li>
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<li> As another control, we'll try 35s-Gal4-H2mRFP </li>
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</ul>
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</div>
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<div id="The plasmid" align="left">
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<h4> End goal plasmid </h4>
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<ul>
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<li> <img src="https://static.igem.org/mediawiki/2014/f/fd/Cambridge-JIC_constructs_GAL4-HAP1GR-H2mRFP.png" width = "100%"> </li>
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</ul>
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</div>
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<h2> Experimentation (up to date)</h2>
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<div id="PCR" align="left">
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<h4>PCR</h4>
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Attempt 1:
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<ul>
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<li>Start date/time: 24/07, 11:08</li>
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<li>End date/time:24/07, 1550</li>
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<li> UIDs of primers/plasmids used:
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<ul>
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<li> A1, P33, P34</li>
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<li> A1, P35, P36</li>
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<li> A2, P37, P38</li>
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<li> A2, P39, P40 </ul></li>
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<li><a href="https://docs.google.com/spreadsheets/d/1RwA7YHMnZ4a0M67RMbegWU5ee3MJRo62qOcBJj537yg/edit#gid=1660015451">PCR Spreadsheet (on google drive)</a></li>
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<li><a href="http://google.com/GEL_27_07_2014">Gel 27/07/2014</a></li>
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</ul>
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</div>
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<div id="Gibson" align="left">
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<h4> Gibson </h4>
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Attempt 1:
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<ul>
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<li>Start date/time: 25/07</li>
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<li>Comments: seemed to be fine.</li>
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</ul>
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</div>
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<div id="E-Coli transformation" align="left">
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<h4>E-Coli transformation </h4>
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Attempt 1:
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<ul>
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<li>Start date/time: 25/07</li>
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<li>Comments:</li>
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</ul> Negative control negative, positive control positive.
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</div>
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<div id="Selection" align="left">
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<h4>Selection </h4>
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Attempt 1:
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<ul>
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<li>Start date/time: 28/07</li>
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<li>Comments:</li>
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</ul> Selected initially just 3 colonies. Grew them in LB and miniprepped on 27/07. Performed digest with SalI to check for template carryover and see that assembly was done. DEXX3, pb>RVe each have a unique site. Following digest with Sal1, we should have a 12kb fragment and a 2kb fragment.
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After digest, 2kb fragment was visible, but 12kb fragment was not. Very mysterious.
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<br>
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<b> Some error in the digestion protocol is likely to have occurred. </b>
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<br>
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To insert a promoter, a digest with Kpn1 was performed. Gel was illegible, although a faint band at 13kb may have been visible. No extraction was made due to poor quality of the gel.
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Attempt 2:
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<ul>
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<li>Start date/time: 30/07</li>
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<li>Comments:</li>
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</ul>
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Grew up in LB 8 more colonies. Attempted colony PCR using inoculation loop in dry PCR tube - will wait to see results from GEL. Expect an 8kb fragment: unlikely that this will show results. Will perform a SalI digest with correct protocol on 31/07 following miniprep from colonies.
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</div>
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<div id="Agrobacteria transformation" align="left">
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<h4>Agrobacteria transformation</h4>
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Attempt 1:
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<ul>
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<li>Start date/time:</li>
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<li>End date/time:</li>
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<li>Induce comments:</li>
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<li>Growth comments:</li>
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<li>Electroporation/selection comments:</li>
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</ul>
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</div>
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<div id="Spore preparation" align="left">
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<h4>Spore preparation</h4>
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Attempt 1:
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<ul>
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<li>Start date/time:</li>
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<li>End date/time:</li>
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<li>Comments:</li>
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</ul>
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</div>
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<div id="Spore transformation" align="left">
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<h4> Spore transformation</h4>
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Attempt 1:
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<ul>
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<li>Start date/time:</li>
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<li>End date/time:</li>
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<li>Comments:</li>
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</ul>
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</div>
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<div id="Evaluation" align="left">
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<h2> Evaluation</h2>
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<ul>
<ul>
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<li><a href="https://2014.igem.org/Team:Cambridge-JIC/Constructs/gal4_hap1gr_H2RBmRFP">Full circuit attempt [SSB]</a></li>
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<li>Start date/time:</li>
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<li>End date/time:</li>
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<li>Comments:</li>
</ul>
</ul>
</div>
</div>
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</html>

Latest revision as of 16:10, 30 July 2014