Team:Cambridge-JIC/Constructs/gal4 hap1gr H2RBmRFP
From 2014.igem.org
(Difference between revisions)
(4 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 50: | Line 55: | ||
Attempt 1: | Attempt 1: | ||
<ul> | <ul> | ||
- | <li>Start date/time: | + | <li>Start date/time: 25/07</li> |
- | + | <li>Comments: seemed to be fine.</li> | |
- | <li>Comments:</li> | + | |
</ul> | </ul> | ||
</div> | </div> | ||
Line 60: | Line 64: | ||
Attempt 1: | Attempt 1: | ||
<ul> | <ul> | ||
- | <li>Start date/time:</li> | + | <li>Start date/time: 25/07</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
Gal4 - Hap1GR - H2BmRFP
Design
Aim of the construct
- We'll try out 35s->GAL4->HAP1GR->H2BmRFP.
- 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
- 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.
- 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
- As another control, we'll try 35s-Gal4-H2mRFP
End goal plasmid
Experimentation (up to date)
PCR
Attempt 1:- Start date/time: 24/07, 11:08
- End date/time:24/07, 1550
- UIDs of primers/plasmids used:
- A1, P33, P34
- A1, P35, P36
- A2, P37, P38
- A2, P39, P40
- PCR Spreadsheet (on google drive)
- Gel 27/07/2014
Gibson
Attempt 1:- Start date/time: 25/07
- Comments: seemed to be fine.
E-Coli transformation
Attempt 1:- Start date/time: 25/07
- Comments:
Selection
Attempt 1:- Start date/time: 28/07
- Comments:
Some error in the digestion protocol is likely to have occurred.
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:
- Start date/time: 30/07
- Comments:
Agrobacteria transformation
Attempt 1:- Start date/time:
- End date/time:
- Induce comments:
- Growth comments:
- Electroporation/selection comments:
Spore preparation
Attempt 1:- Start date/time:
- End date/time:
- Comments:
Spore transformation
Attempt 1:- Start date/time:
- End date/time:
- Comments:
Evaluation
- Start date/time:
- End date/time:
- Comments: