Rahul Palnitkar, Week 6: One Step Forward, Three Steps Back
This past week was one of both successes and failures. On the one hand, my Co-immunoprecipitation experiment went well, and we were able to establish a link between Prdm16 and p53. On the other, we had a spate of bad luck concerning transformation and plasmids.
The week started off fairly normally. I spent that Monday preparing and running my protein gel. I found out rather quickly that protein gels were nothing like agarose-based gels; protein gels were much more difficult to load precisely, and the gel had to be handled with the utmost care. While the gel was running, I busy preparing a presentation I would give during lab meeting the next day, and I was having quite a bit of trouble. I didn't quite know what to say, and I had no results I could show in time for lab meeting. Finally, I settled on explaining what I was doing, why I was doing it, and expected results (almost like the spring term EXP poster, in a way).
Thankfully, I was not going to be the sole presenter, as two undergrads also had to present during that lab meeting. The meeting actually went quite well, and there was a lot of great discussion at the meeting. Later that same day, we finished the co-immunoprecipitation experiment, which showed us that Prdm16 does interact with p53. All in all, Tuesday was quite a successful day.
The same could not be said of the rest of the week. First, the plasmids we were finally able to transform into bacteria came back from sequencing, and it turned out that instead transforming the modified plasmid, we had transformed the original, indicating that there was still some uncut pure plasmid that had accidentally been transformed into the bacteria. We tried to start over and digest some new plasmid, but that was when we ran into our second problem. We found out that one of our restriction enzymes had been cutting in two places on the plasmid rather than one, meaning that there was a hidden restriction site on our plasmid, which in turn rendered Gibson assembly useless.
We tried solving our plasmid problem using two different approaches. First, I was to do a partial digest of the plasmid and see if that solved our problems. We didn't expect this method would work, but it was worth a shot. However, this method failed, because I apparently forgot to put any plasmid in the plasmid digest. The second approach was to use different primers for a different restriction site, but it would take a week or so before they could arrive.
All in all, the week was undoubtedly exciting, although I'm still quite annoyed that I forgot to put the plasmid into the plasmid digest.
The week started off fairly normally. I spent that Monday preparing and running my protein gel. I found out rather quickly that protein gels were nothing like agarose-based gels; protein gels were much more difficult to load precisely, and the gel had to be handled with the utmost care. While the gel was running, I busy preparing a presentation I would give during lab meeting the next day, and I was having quite a bit of trouble. I didn't quite know what to say, and I had no results I could show in time for lab meeting. Finally, I settled on explaining what I was doing, why I was doing it, and expected results (almost like the spring term EXP poster, in a way).
Thankfully, I was not going to be the sole presenter, as two undergrads also had to present during that lab meeting. The meeting actually went quite well, and there was a lot of great discussion at the meeting. Later that same day, we finished the co-immunoprecipitation experiment, which showed us that Prdm16 does interact with p53. All in all, Tuesday was quite a successful day.
The same could not be said of the rest of the week. First, the plasmids we were finally able to transform into bacteria came back from sequencing, and it turned out that instead transforming the modified plasmid, we had transformed the original, indicating that there was still some uncut pure plasmid that had accidentally been transformed into the bacteria. We tried to start over and digest some new plasmid, but that was when we ran into our second problem. We found out that one of our restriction enzymes had been cutting in two places on the plasmid rather than one, meaning that there was a hidden restriction site on our plasmid, which in turn rendered Gibson assembly useless.
We tried solving our plasmid problem using two different approaches. First, I was to do a partial digest of the plasmid and see if that solved our problems. We didn't expect this method would work, but it was worth a shot. However, this method failed, because I apparently forgot to put any plasmid in the plasmid digest. The second approach was to use different primers for a different restriction site, but it would take a week or so before they could arrive.
All in all, the week was undoubtedly exciting, although I'm still quite annoyed that I forgot to put the plasmid into the plasmid digest.
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