I think I lost count of rotation weeks somewhere back there... for consistency, I'll call this Week 7, but I actually have two more weeks left. Admission to PhD program in neuroscience? Check. Counting to single-digit numbers? Fail.
The sub-cloning project continues. I had one restriction enzyme digest that didn't work, so we chose a new enzyme for those samples and seemed to get it on the second try. I cut and blunted and cut and gel-purified, in between studying for my final exam and finishing my group project. These experiments lend themselves well to a crazed finals week, as I could set up a lot of reactions in an hour of lab time and just let them go overnight. Running the gels takes a while, though, as I'm working with some pretty big chunks of DNA and they are slow to separate, even on a low-density agarose gel. Outside the lab, I was proud of myself for coming up with a study schedule and sticking to it. I outlined all of the lectures, made flash cards, and got together with classmates to review material, without last-minute cramming. After taking the test on Thursday, I felt pretty good about it (although one professor did ask some really picky questions... we'll see).
I'm still waiting on the sequencing results from the mystery mice. Actually, I haven't even sent the samples to the sequencing facility yet. For some reason it's taking a long time to get purchases approved by the person managing the grant that funds these experiments, so every time I want to get something sequenced, there's a turnaround time of sometimes three days before I get the purchase order. By the time we got the funds approved for this batch, it was Friday, and no one at the sequencing facility would be there to receive our samples on Saturday, so they'll sit in the freezer this weekend and go out on Monday. One solution to this issue would be to put in a request for a really huge purchase order and reuse it many times over a couple of months, but you're technically not supposed to do this. You're just supposed to wait. Red tape is everywhere, even in the lab. Oh well.
Practiced some more stereotaxic surgeries this week. The concept seems pretty simple: anesthetize the animal, stabilize the head in the stereotax, do the procedure (in this case, we expose the skull, drill a tiny hole in it, and insert a needle into the brain to deliver our treatment), close the animal up, and let it recover (including medication for any post-operative pain). In practice, it's hard to get the head position right and adjust the arm that holds the needle/syringe into the right spot. I was constantly worried that I would do something wrong and hurt the mouse, although it was under anesthesia and felt no pain. We didn't do any survival surgeries; we sacrificed the animals immediately after injection and dissected them to check that the injection site was in the right place. Although this doesn't seem very nice to the mice, it's important to optimize the procedure before conducting the real experiments, to ensure consistent and meaningful results. This cuts down on animal use, overall. The dissection looked pretty good, so this weekend a postdoc from the lab will be doing the real experiment with some Cre virus. Once the animals recover and the virus has a chance to do its thing, we could see some interesting results. Probably this will take more time than I have left in my rotation, but I feel invested enough in it that I want to know what happens.
Although I'm just a trainee, I felt useful this week. A labmate presented some data a while ago and mentioned a weird result that might indicate a flaw with either the transgenic mouse line or with the fundamental assumption behind the experiment. When this happens, we tend to assume a technical problem first -- the mouse isn't what you think he is. Checking on this is difficult, though, because we don't have good antibodies for the protein this mouse is supposed to lack, and it's a conditional knockout, so some of its cells do have the gene and some don't (making it hard to just isolate mRNA and look for gene expression at that level). In situ hybridizations to look at the gene expression would work, but it can be hard to look at individual cells this way. Anyway, I read some papers for my last rotation where a group used laser microdissection to isolate individual cells from a brain slice, purified RNA, and then did RT-PCR to figure out if the cells were expressing certain genes of interest. I looked around and saw that Emory has a core facility that will do laser microdissection for a reasonable rate, or train lab members in the technique so they can do it themselves. We can already do RT-PCR in the lab, too. So I suggested that we try that, rather than in situs, and my labmate seemed really into the idea. I think he was already exploring this possibility with our PI, but they didn't know about the core facility. So I felt all smart and helpful for mentioning it. It's been cool to realize just how much I'm learning in grad school, and that I'm coming up with good ideas for experiments on my own. My education is working!
Now that finals are done, I have a lot of free time in my schedule for these last few rotation weeks. Then I'll be presenting my results in lab meeting, writing up a report, and choosing which lab I want to join. I might take a little summer vacation first, though...
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