Team:WashU/Week5

YLC

At the end of last week we ran a gel of the digests from the fluorescent proteins and extracted DNA from the gels. At the beginning of this week, we used the nanodrop to see how much DNA we had retrieved from the gel. The nanodrop revealed barely imperceptible maxima to indicate that we had almost undetectable amounts of DNA by the nanodrop. In fairness, the bands were tiny to begin with, leading to our poor results. Our mentors assured us that we probably had enough DNA for a ligation, so we plan to run that once the promoter digest works.

We accidentally grew up the new promoter, J23119, in both plain LB and LB + amp, yet still had colonies in both media, and thus decided to run two digests to ascertain that we have our promoter in both cultures. We used the biobrick protocol with a slight modification - we used NEBuffer 4 instead of NEBuffer 2, since the enzymes we were cutting with, E and S, also have 100% activity in NEBuffer 4 - and ran a gel to ensure that we had pure promoter. The gel is shown below:

Unfortunately, the digest did not seem to work. We attempted to digest the same DNA once more, using NEBuffer 2 instead of NEBuffer 4, and ran a second gel. This gel also reveals that the digest was unsuccessful, as we see only one band when we expect to find two bands. We believe that our enzyme SpeI has not been cutting properly.

To test what was going wrong, we decided to run one final gel. We digested our promoter, just like before, but used four different combinations of enzymes. We digested with EcoRI and SpeI (ES), XbaI and SpeI (XS), EcoRI and PstI (EP), and XbaI and PstI(XP). This gel was also a failure, as we only saw one band for each well, when we should have seen two.

We have also decided to use PCR to amplify our DNA, as we are getting extremely low yields from our gel extractions. Thus, we designed the primers today.

Website

The weekly logs were revised and updated today.

Tuesday, June 26

YLC/Saffron in a Kan

We decided to figure out what was going wrong with our digests by running two new gels, one for the E. coli part of the project and one for the YLC project. First, we used the nanodrop to measure how much DNA we had for each of our samples. The data is shown below.

Sample	nanograms/microliter
1: YFP	83.0
2: GFP	121.6
3: mRFP	63.8
4 - I: eCFP	83.9
4 - II: eCFP	99.4
5: promoter BBa_J23100	127.0
8: mCherry	183.4
9: carotenoids inE. coli	84.1
10: crtZ inE. coli	37.5
Promoter J23119	34.5

Then, we ran digests of 9, 10, 5 and BBa J22119 using controls, DNA cut with the enzymes as stated in the biobrick assembly protocol(shown with a "c" below), and then DNA cut with enzymes and then treated with phosphatase (shown as P- on the gel). For the YLC project, we ran 1, 2, 3, 4 and 8. [Gels shown below]



Our digests appear to be successful this time. We extracted the DNA for all of the YLC runs except for 4 and also extracted 9 from the first gel, and then purified all of the above bands.

YLC

We finally ordered the primers from Sigma today and they will arrive in a few days.

Wednesday, June 27

YLC/Saffron in a Kan

After our meeting with our mentor Dr. Dantas this morning, we decided to try a ligation.

Thursday, June 28

Friday, June 29

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