This was a BIG week for me in the lab. Last Friday, I was FINALLY able to amplify a gene that seems to be approximately the length of alas2!!!!!!!!!!!!!! I had been going through all the primers I had designed before break and re-evaluating them based on if they had been created specifically for gDNA (ie. working around an intron, bridging the UTR and flanking sequences, etc.), and re-locating the ones I had created for cDNA in the gene sequence. Mine and Larissa’s latest theory had been that the cDNA I was using in the beginning of the year was defective in some way, either because the embryos had not been expressing alas2 transcripts when it was made, or because it had gotten partially degraded, or something along those lines. After I made new cDNA, I had been trying various primers and temperatures and using housekeeping genes to ensure that I had created cDNA from the RNA of the embryos in the first place. The magical combination for alas2 turned out to be……Forward 3 and Reverse 1 at 50˚C!!!!!! Woohoo. I sent the PCR products for sequencing so will hopefully hear back from that lab this week, and can confirm if I got something that is close to alas2.
I also learned to microinject last week! In the weeks before that, Mel had been teaching me the setup and steps of injecting, and got me accustomed to checking the gas tank, pulling needles, loading needles, etc. Last week was my first time actually staggering barrier pulling, collecting embryos to inject, setting up the machine, and getting some practice in. Turns out shearing the needles and setting up the apparatus so that it inserts into the embryos just right is super hard!! I am going to practice with Mel more tomorrow.
In other new, the rest of the lab is also doing big things. Mel has been injecting Nfe2 morpholino into embryos and measuring their ears, and then taking confocal pics of their neuromasts in the otic vesicle. Her pics are pretty and sometimes she lets me look in the microscope in lab. She is also the best microinjecting teacher ever. Gwen has been doing lots of ChIP, and that’s what the article she chose for us this week is on. She is mainly working out the immunoprecipitation protocol, and is killing the game!! Jason is doing microinjecting on microinjecting—he moved his main day to Thursday, so he is next to me right now injecting Nfe2 morpholino and after will be scoring as per usual. He is super good at microinjecting too but I’m thinking I can challenge his skills once I get better (kidding Jason you rock).
Also, last week, Gwen and I jumped in the puddle. It was super cold. We are seniors and it’s weird.