I seem to have missed a few rotation diaries -- sorry about that! Things are getting more and more hectic as the semester winds down. I had a big exam at the end of March, I've got some projects in the works for my graduate seminar, and for the past week I've also been fighting a losing battle against housework while tending to my partner, who has come down with the flu. Even so, I had time to do some things for my rotation.
My latest project has been to assist a postdoc in the lab with some sub-cloning. This I would describe as the Legos of biology -- you have all these pieces that need to fit together to make the thing that you want, and a set of rules for how the pieces can be swapped out and combined with each other. Instead of plastic bricks, we use pieces of DNA, so the end result is harder to show off to people. I've still been showing off pictures of my gels, though.
We started with some DNA plasmids that the lab already had. The goal is to snip out some useful pieces of DNA and put them into other plasmids that will be used to make a lentivirus. Then we'll be able to infect cells with the virus and make them express the genes we're interested in. (One construct is a YFP-Cre, which will allow us to knock out a gene of interest in floxed mouse brain cells near the viral injection site through Cre-lox recombination, without messing with the rest of the brain. Nifty!)
To make these new constructs, I've had to digest the DNA with a bunch of different restriction enzymes, which cut DNA in predictable places. I've also been analyzing the sequences of the plasmids, to make sure that nothing funky happened to them when we grew them up in bacteria to get the quantity of DNA that we need. So far it's been pretty good, although I had a false start with my first digestion (I made a "transcription error" in copying the postdoc's instructions into my notebook, and left out an important reagent). I also got a much lower yield than I expected after I gel-purified my digested DNA. I don't know what happened, there, since I just used a commercial kit for that step, and those things are fairly idiot-proof. I may just be an unusually talented idiot.
Currently scrambling to finish a poster for grad seminar (needs to be sent to the printer's on Monday; I finally gave a draft to my professor this afternoon...). It's my first scientific poster (not counting elementary school creations that featured a lot of construction paper), and I'm amazed at how time-consuming it can be to take things that you already have or know and put them into a new format. I spent forever tinkering with background colors and fonts and things, seeking clarity. Hopefully it'll come out all right. I put off working on it for much longer than I should have (partly my own procrastination, partly the unpredictability of life), so my goal at this point is to get it done rather than make it perfect.
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