Week of 7/16/07 - 7/20/07
Tests and Modeling:
With the modeling finally understood, we begin design REAL testing constructs that could determine values for constants in the model.
1. Alpha values (promoter and RBS strengths)
2. Beta values (repressor cooperativity)
3. Inducer Strength
We made some preliminary tests and showed them to Jason who helped us to refine them a bit further. In response, the ligation plans database was also changed to accommodate these new constructs.
Restriction Digests for Phase I of tri-stable ligations were gel extracted, ligated, and transformed.
- Gel turned out well: still problems with ladder but DNA bands were very clear and exactly where they should have been.
- Gel Extractions were not as good. Eluting DNA in 50 ul gives ridiculously low concentrations (around 3ng/ul). These were concentrated through ethanol precipitation in 4ul of water and concentration subsequently around ~90ng/ul.
- Transformations: because the plasmid uptake efficiency of our competent cells are still rather sketchy, it remains to be seen next week if these transformations will actually turn out.
In other news, Norris was working on 3A ligations this week. The first attempt did not transform (only 2 colonies) but this may have been due to weak batch of competent cells - other people on the team were having problems with this as well. Kyle has made a new batch and a second attempt will be tried next week. Other considerations:
- ccDB on psb1AK3 (vector) was at a low concentration after PCR purification. Ethanol precips seem to work much better. - we used 3 ul of vector and only 1-2 ul of each insert.
The two colonies were cultured anyway and geled. From gels, they seemed to have taken up the plasmid anyway. Digested DNA was geled Friday evening and the plasmid was the right size. However the part did not show up - this may be due to not enough DNA being used. Will try again next week.
In other other news. Finally found the perfect recipe for DNA ladders! We were using too much DNA. Recipe was:
.5 ul ladder
2 ul 10X Blue Juice
8 ul ddH20