Team:Cambridge-JIC/Constructs/gal4 hap1gr H2RBmRFP

From 2014.igem.org

(Difference between revisions)
 
(5 intermediate revisions not shown)
Line 1: Line 1:
 +
{{:Team:Cambridge-JIC/Templates/header_prototype2}}
 +
<html>
<html>
 +
 +
<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>
 +
<h1> Gal4 - Hap1GR - H2BmRFP</h1>
<h1> Gal4 - Hap1GR - H2BmRFP</h1>
Line 7: Line 12:
<ul>  
<ul>  
<li> We'll try out 35s->GAL4->HAP1GR->H2BmRFP. </li>
<li> We'll try out 35s->GAL4->HAP1GR->H2BmRFP. </li>
-
<li> Since H2BmRFP has not been tried in Mp, we'll also try 35s->H2BmRFP </li>
+
<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>
 +
<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.
 +
<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>
 +
<li> As another control, we'll try 35s-Gal4-H2mRFP </li>
</ul>
</ul>
Line 47: Line 55:
Attempt 1:
Attempt 1:
<ul>
<ul>
-
<li>Start date/time:</li>
+
<li>Start date/time: 25/07</li>
-
<li>End date/time:</li>
+
<li>Comments: seemed to be fine.</li>
-
<li>Comments:</li>
+
</ul>
</ul>
</div>
</div>
Line 57: Line 64:
Attempt 1:
Attempt 1:
<ul>
<ul>
-
<li>Start date/time:</li>
+
<li>Start date/time: 25/07</li>
-
<li>End date/time:</li>
+
 
<li>Comments:</li>
<li>Comments:</li>
-
</ul>
+
</ul> Negative control negative, positive control positive.
 +
</div>
 +
 
 +
<div id="Selection" align="left">
 +
<h4>Selection </h4>
 +
Attempt 1:
 +
<ul>
 +
<li>Start date/time: 28/07</li>
 +
 
 +
<li>Comments:</li>
 +
</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.
 +
 
 +
After digest, 2kb fragment was visible, but 12kb fragment was not. Very mysterious.
 +
<br>
 +
<b> Some error in the digestion protocol is likely to have occurred. </b>
 +
<br>
 +
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.
 +
 
 +
Attempt 2:
 +
<ul>
 +
<li>Start date/time: 30/07</li>
 +
 
 +
<li>Comments:</li>
 +
</ul>
 +
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.
</div>
</div>

Latest revision as of 16:10, 30 July 2014