From 2010.igem.org
(Difference between revisions)
|
|
Line 59: |
Line 59: |
| </table><p><br /> | | </table><p><br /> |
| | | |
- | **Click on the individual pieces to view how we ligated them together.** <p><br /> | + | **Click on the individual pieces above to view how we ligated them together.** <p><br /> |
| + | |
| + | <a name="plasmid"></a><p class="header"><b>Placing them into individual plasmids</b></a><p> |
| + | <ul> |
| + | <li>After we have created the individual pieces, we must ligate the composite parts together into a total of two plasmids. </li> |
| + | </ul><p><br /> |
| + | |
| + | <img src="https://static.igem.org/mediawiki/2010/3/36/Plasmid1.jpg"><br /> |
| + | <img src="https://static.igem.org/mediawiki/2010/1/12/Plasmid2.jpg"><p><br /> |
| | | |
| | | |
Revision as of 21:01, 27 October 2010
|
|
|
**Click on the individual pieces above to view how we ligated them together.**
- After we have created the individual pieces, we must ligate the composite parts together into a total of two plasmids.
![](https://static.igem.org/mediawiki/2010/3/36/Plasmid1.jpg)
![](https://static.igem.org/mediawiki/2010/1/12/Plasmid2.jpg)
![](https://static.igem.org/mediawiki/2010/2/29/Initiatorconstruct.jpg)
Because the initiator is one of the smaller constructs, we have decided to use this as an example to illustrate the utmost basic workplan in greater detail.
Starting Up
- Hydrated parts: BBa_B0034, BBa_C0051, BBa_B0015, BBa_R0082.
- Transformed these parts into DH5α competent cells.
RBS to BBa_C0051
- Ligate the RBS and BBa_C0051 fragments together and transform on carb plates.
- Depending on the outcome of visible colonies on the experimental and control plates, a PCR screening may be needed.
![](https://static.igem.org/mediawiki/2010/b/bb/RBSc0051.jpg)
Assembly notes
- It is most practical to cut RBS as a vector because RBS is less than 100 bp long as an insert, making it hard to see and extract on the gel.
- The sticky ends from the XbaI and SpeI, when ligated together, will form an 8 bp scar. Thus, XbaI and SpeI will no longer be able to cut here.
'RBS & BBa_C0051' to Terminator
- Cultured cells that have parts: 'RBS & BBa_C0051', BBa_B0015
- Miniprepped 'RBS & BBa_C0051' and BBa_B0015
- Ligate the fragments together and transform on kan plates.
- Depending on the outcome of visible colonies on the experimental and control plates, a PCR screening may be needed.
![](https://static.igem.org/mediawiki/2010/e/eb/RBSc0051stop.jpg)
Assembly notes
- It is most practical to cut BBa_B0015 as the vector because it is only 130 bp long. It would be hard to see on a gel.
'RBS & BBa_C0051 & stop' to BBa_R0082
- Cultured cells that have parts: 'RBS & BBa_C0051 & stop', BBa_R0082
- Miniprepped 'RBS & BBa_C0051& stop' and BBa_R0082
- Ligate the fragments together and transform on carb plates.
- Depending on the outcome of visible colonies on the experimental and control plates, a PCR screening may be needed.
![](https://static.igem.org/mediawiki/2010/3/34/Initiatorplasmid.jpg)
Assembly notes
- The promoter had to be cut as a vector because it is only 108 bp by itself.
|
|
|
|
|
|
![](/wiki/images/0/00/Sponsors.jpg) |
We would like to take a moment to thank all of our sponsors for their very generous donations, as we could not have done this without your help!
![](/wiki/images/a/a4/Rcsponsor.jpg)
![](/wiki/images/6/6e/Cschwartzsponsor.jpg)
![](/wiki/images/f/fe/Fisherscientific.jpg)
![](/wiki/images/c/c6/Irtacal.jpg)
![](/wiki/images/4/49/Ucdce.jpg)
![](/wiki/images/3/3f/Ucdgenome.jpg)
![](/wiki/images/d/df/Wssponsor.jpg)
![](/wiki/images/d/d6/Dcsponsor.jpg)
![](/wiki/images/5/52/Cfremaxgold3.jpg)
![](https://static.igem.org/mediawiki/2010/9/98/Ansponsor.jpg)
We would also like to thank and acknowledge:
Our Advisors
Marc Facciotti
Ilias Tagkopoulos
Technical Guidance
David Larsen
Andrew Yao
Visiting iGEMer
Jia Li of Zhejiang University (TEAM ZJU-China)
cI Promoter Screen
Drew Endy - Stanford
Thomas Schneider - NIH
Want to sponsor us? Send an email to mtfacciotti@ucdavis.edu to discuss various ways you can help! :)
|
|
|