"
Team:Technion/11 September 2012
From 2012.igem.org
(Difference between revisions)
| Line 7: | Line 7: | ||
- Tested ladders on a 0.6% agarose gel. Ran for an hour and 10 mins at 40V on a minigel. The 1Kb opened but the high range (HR) ladder didn't open. We will use a 0.4% agarose gel in the future attempts.<br> | - Tested ladders on a 0.6% agarose gel. Ran for an hour and 10 mins at 40V on a minigel. The 1Kb opened but the high range (HR) ladder didn't open. We will use a 0.4% agarose gel in the future attempts.<br> | ||
- Hila and I did the first attempt for gibson on the phage fragments. We attempted an assembly of fragments #4 (6150bp) and #5 (8080bp). The results are presented in the following gel pic: <br> | - Hila and I did the first attempt for gibson on the phage fragments. We attempted an assembly of fragments #4 (6150bp) and #5 (8080bp). The results are presented in the following gel pic: <br> | ||
| - | + | [[File:Two parts together 11.9.12.jpg|350px]]<br> | |
As you can see in the attached file, only one of our three attempts today actually worked. The reaction conditions for the attempt that worked were: 100bp overlaps, ~150ng DNA of each of the templates (~0.025-0.03 pmoles), 5ul of Gibson mix and 5 ul of DNA fragments (10ul final volume). All reactions were incubated for 1 hr at 50 degrees as instructed by the manual.<br> | As you can see in the attached file, only one of our three attempts today actually worked. The reaction conditions for the attempt that worked were: 100bp overlaps, ~150ng DNA of each of the templates (~0.025-0.03 pmoles), 5ul of Gibson mix and 5 ul of DNA fragments (10ul final volume). All reactions were incubated for 1 hr at 50 degrees as instructed by the manual.<br> | ||
I think that the 20 ul final volume reaction with the 100bp didn't work because there was too much DNA. The 400bp overlap might still work with additional exonuclease and in a 10ul volume with ~150ng of each fragment. | I think that the 20 ul final volume reaction with the 100bp didn't work because there was too much DNA. The 400bp overlap might still work with additional exonuclease and in a 10ul volume with ~150ng of each fragment. | ||
Revision as of 05:18, 12 September 2012
- Home
- Team
- Project
- Parts
- Notebook
- Safety
- Attributions & Achievements
- Human Practices
- Sponsors & Thanks
|
|
|
|
|
|
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Ilya
- Minipreped the picked colonies for testing the clones of Fus2 and Fus2del in pCP.
- Did a restriciton digest with BamHI-HF to test the colonies for the correct insert. 3/3 clones of Fus2del and 3/4 clones of Fus2 were positive. I picked one of each of the clones for plate reader tomorrow.
- Put starters for plate reader tomorrow.
- Tested ladders on a 0.6% agarose gel. Ran for an hour and 10 mins at 40V on a minigel. The 1Kb opened but the high range (HR) ladder didn't open. We will use a 0.4% agarose gel in the future attempts.
- Hila and I did the first attempt for gibson on the phage fragments. We attempted an assembly of fragments #4 (6150bp) and #5 (8080bp). The results are presented in the following gel pic:
As you can see in the attached file, only one of our three attempts today actually worked. The reaction conditions for the attempt that worked were: 100bp overlaps, ~150ng DNA of each of the templates (~0.025-0.03 pmoles), 5ul of Gibson mix and 5 ul of DNA fragments (10ul final volume). All reactions were incubated for 1 hr at 50 degrees as instructed by the manual.
I think that the 20 ul final volume reaction with the 100bp didn't work because there was too much DNA. The 400bp overlap might still work with additional exonuclease and in a 10ul volume with ~150ng of each fragment.
