Team:Cambridge-JIC/Constructs/gal4 hap1gr H2RBmRFP

From 2014.igem.org

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Attempt 1:
Attempt 1:
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<li>Start date/time:</li>
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<li>Start date/time: 25/07</li>
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<li>End date/time:</li>
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<li>Comments: seemed to be fine.</li>
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<li>Comments:</li>
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Attempt 1:
Attempt 1:
<ul>
<ul>
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<li>Start date/time:</li>
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<li>Start date/time: 25/07</li>
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<li>End date/time:</li>
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<li>Comments:</li>
<li>Comments:</li>
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</ul> Negative control negative, positive control positive.
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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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<b> Some error in the digestion protocol is likely to have occurred. </b>
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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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Latest revision as of 16:10, 30 July 2014