The Ups and Downs of Research: A Real Rollercoaster Ride
The past week in lab has been busy as usual.
Since getting back from break, I’ve been collecting and treating embryos with tBOOH at different time points to try to drive Nfe2 expression. I finished collecting several time points I am interested in this past weekend and was planning to do a Western on them this week to probe for Nfe2 in the samples. However, the gel did not run straight. This was really disappointing and frustrating, as I thought I worked out these problems before break. After some troubleshooting and with Larissa’s help, we think we figured out the problem (we were trying to run too many samples) and I got a gel to run straight by the end of the day. I wasn’t able to do the full Western, but I did transfer it to nitrocellulose paper and did a Ponceau stain to look for proteins. It was encouraging that both samples I ran had a good amount of protein in them!
I have also been practicing the first few steps of ChIP over the past couple of weeks, as I will be moving on to it soon to hopefully figure out where Nfe2 is binding. I cross-linked the proteins to DNA in 24 hpf embryos last week using formaldehyde and then I sonicated the sample this week to break the DNA into 200 bp fragments. The sonication was successful, which is great because now I can move on to immunoprecipitation. I love the new sonicating bath that makes the process really simple and easy!
My fellow lab members have been hard at work as well. Rachel is doing PCR with more cDNA that she made and rechecking primers because some were made specifically for gDNA and now they won’t work. She really is a PCR queen! Jason is continuing to inject and score. Mel is also injecting and scoring the ears, and she is working out the kinks on the confocal to get pictures of the neuromasts in the otic vesicle. Lastly, Mel taught Rachel how to inject, so that’s exciting! I really enjoy Tuesdays when we are all in the lab together for at least a little while. It’s so fun to have other people around to talk to about how exciting (and frustrating) science can be!