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, XbaI and SpeI, EcoRI and PstI, and XbaI and PstI. 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.
The weekly logs were revised and updated today.
Tuesday, June 26
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.
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, and then DNA cut with enzymes and then treated with phosphatase. For the YLC project, we ran 1, 2, 3, 4 and 8. [Gels shown below]
We finally ordered the primers from Sigma today and they will arrive in a few days.
Wednesday, June 27
Thursday, June 28
Friday, June 29
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