Rahul Palnitkar--Weeks 4 and 5
So these past two weeks were extremely busy, and quite eventful as well. Most of the week was spent trying to transform DH5a competent cells with specific Prdm16 plasmid we made using Gibson Assembly. We tried increasing the concentration of plasmid, using new Gibson Assembly master mix, and even tried to run a positive control to make sure everything was working. All of our attempts failed, indicating either the cells or the master mix was the problem. So I transformed pure plasmid into the DH5a cells to see if these would transform, and they did...sort of. There were indeed bacterial colonies, but our transformation efficiency was 100x lower than what it should have been. This obviously was a problem, so we ordered new, ultra-competent bacteria to see if we could at least transform those. And indeed, our Gibson Assembly worked.
All total, we spent about one-and-a-half weeks just trying to get the transformations to work. However, that was not the only project we did. In addition to bacterial transformation, I also continued to passage my HCT116 cancer cells, plated new Caco2 cancer cells, and induced apoptosis in the organoids I grew a few weeks ago.
As you can see above, the wild type organoids look healthy and normal, while the Prdm16 KO organoids look quite dead. This was the phenotype we hoped to achieve, so this project was a success.
Finally, I transfected my HCT116 cells with Prdm16 and p53 and ran a co-immunoprecipitation to see if Prdm16 interacted with p53 in small intestine cells. This project was by far the most involved one I had done at the lab, and it was quite time consuming too; the project was spread over four days. First, I washed and harvested the cells, and then I transfected them with lipofectamine and my plasmid. Lipofectamine is a lipid based substance that essentially envelopes the plasmid DNA and allows it to pass through the phospholipid cell membrane. After a day or so, I began the actual process of co-immunoprecipitation. I harvested the proteins, mixed them in with flag beads containing antibodies, and allowed the reaction to take place. I haven't run a western blot on the proteins yet, although that is the next step.
All total, we spent about one-and-a-half weeks just trying to get the transformations to work. However, that was not the only project we did. In addition to bacterial transformation, I also continued to passage my HCT116 cancer cells, plated new Caco2 cancer cells, and induced apoptosis in the organoids I grew a few weeks ago.
 |
| Wild Type Organoids (2 Days after 4OHT treatment) |
 |
| Prdm16 KO Organoids (2 Days after 4OHT treatment) |
As you can see above, the wild type organoids look healthy and normal, while the Prdm16 KO organoids look quite dead. This was the phenotype we hoped to achieve, so this project was a success.
Finally, I transfected my HCT116 cells with Prdm16 and p53 and ran a co-immunoprecipitation to see if Prdm16 interacted with p53 in small intestine cells. This project was by far the most involved one I had done at the lab, and it was quite time consuming too; the project was spread over four days. First, I washed and harvested the cells, and then I transfected them with lipofectamine and my plasmid. Lipofectamine is a lipid based substance that essentially envelopes the plasmid DNA and allows it to pass through the phospholipid cell membrane. After a day or so, I began the actual process of co-immunoprecipitation. I harvested the proteins, mixed them in with flag beads containing antibodies, and allowed the reaction to take place. I haven't run a western blot on the proteins yet, although that is the next step.
Comments
Post a Comment