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Sarah, Week 5-6

I began week 5 by using the microscope to view the cell plates that I had previously stained.  It was really exciting both to have results and to also see that I had followed the procedure correctly.  I was a little bit nervous that something that gone wrong along the way and that the microscope but be unable to see the neurons, but I was able to view the red, blue, and green stains.  I then had to set points in the center of each well and then focus the image and change the brightness, in order for the microscope to, hopefully, capture.  However, I was a little unlucky and had to keep restarting the process because the microscope kept crashing, and was not able to take the 25 images needed of each well.  Instead,  Claire was able to take a single image of each well by zooming out, leading to a larger picture.  We were happy to find that the results were as expected, with cell death occurring in wells in which it was predicted, and the cells in the vehicle control remaining healthy in comparison.
The next task was to create more Western blots.  I began by doing the Bradford in order to determine the dilutions necessary for each sample.  After having done it before, it was a lot easier for me to move quickly through analyzing each cuvette in the nanodropper, so that I would have the best possible results.  Upon analyzing the protein concentration in each sample, I was then able to do dilutions with the sample, adding in a purple loading buffer.  The next day, after running two blots, I then had to transfer them onto a membrane.  For transfers and running, the lab has variety of different sized transfer units.  My favorite for transfers is the midi, which looks like the gel, membrane, filter paper, and sponge sandwich is squeezed into a connect four.  Because of the large number of gels being run, I also needed to use the mini gel tanks, which the lab is just beginning to figure out how to use.  I was a little bit worried because other people had had issues with their gels melting in the mini while transferring due to it overheating.  So, the while most of the gels were all set to transfer overnight, the gels in the mini were set to transfer in a few hours.  This was so that it would be part of a trial and error to determine how long and how much voltage could be used before the gel became melty.  The bottom of the gel did slightly melt, but, luckily, not where the protein was predicted to be.
I then worked on extracting DNA from tails and genotyping mice using gel electrophoresis.  Upon imaging the gel, Claire was very excited to find that she had her first wild type mouse!  I was really happy to be a part of this because it's the small achievements that matter.  After having bred so many homozygous and heterozygous mice, it was so exciting to finally have a wild type mouse.
As my last day is August 3rd, and Claire has a thesis committee on July 31st, we are beginning to quantify the Western blots and to put together data.  6 weeks have gone by so quickly, so I am glad that I have two more weeks left to continue working in the lab.
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