March Progress (and some madness)
We are about a month out from binding our final theses (crazy!) and everyone is hard at work in lab and Ladd.
I have made a lot of progress with my project over the past few weeks and am finally able to begin immunoprecipitation with Nfe2! Over break, I got a positive control with a histone antibody to work, indicating that the immunoprecipitation was successful. I was really happy that I got this new protocol to work on just the second go around because it involves a lot of steps and there are many points at which it could go wrong. Although the immunoprecipitation worked, I had difficulty amplifying the input with qPCR, but we figured out this week that there was actually too much DNA (a good problem to have) and it worked after I used less DNA. I also designed primers last week around supposed Nfe2 binding sites so we can do qPCR after I immunoprecipitate with Nfe2. It was cool, and a bit frustrating, to get a taste of primer design and what Rachel has been doing this whole year.
My primer pairs worked in both PCR and qPCR this week, which was awesome, and now we can start immunoprecipitation with Nfe2. This is essentially the climax of my project and all my hard work for the past few months has been building to this, so I’m really excited!
Everyone else has been hard at work as well. Mel is going to take more pictures of Brn3c embryos injected with nfe2 MO on the confocal to get some more data and is also going to dose some with tBOOH this weekend and image those on the confocal as well. Jason has been injecting and scoring and is excited to be done with the scoring phase next week. Rachel has been getting better success rates with injecting the control MO, and she is also doing more PCR, trying new primers and different conditions. Larissa is off to a conference this coming week and was working on a poster for it. She is presenting the data on the Ahr ChIP project along with Alex and Jake, some past thesis students.
Lastly, we have all been working on writing our results and discussion and adding to our methods, as we have a draft due tomorrow. It’s been a lot of work and I think we are all a bit tired, but it is really exciting that we’re all making progress in the lab and on paper!