Week Three

As I thought, I worked much more hands-on this week. For Monday and Tuesday, it was only the graduate student and I, so I took on some of the responsibilities that normally would be the lab tech's, including helping take care of the mice instead of just observing. Later in the week, I was sharing some of these responsibilities with the other undergraduate student. For the most part, the tasks were PCRs and gels, however, these were some of the first DNA analyses we were able to do that were relevant to the research and not just for practice. Our results tell us details about the genotype of the mice we work with, and all of the genotyping information must be kept in the same notebook as a reference for deciding how to use the mice. Though the PCRs and gels are fairly short and straightforward to run, their accuracy is necessary and they need to be completed well every time.

With new processes I have started, I worked on cells that allowed me to practice. I believe this is best because it is easy to make small errors the first time doing new techniques, and mistakes can be detrimental when they cause lengthy processes to have to be redone or for expensive or rare samples to be wasted. I had a natural ability for some of the new techniques and was able to complete them correctly the first time I tried; others I need more practice with.

The cells I received to work on were HEK 293 cells, which are human embryonic kidney cells. When I first saw them, I was intrigued by their shape. I have never worked with cell cultures using a liquid medium, so I had to learn all of the small details that keep them and alive and growing strong. In general, bubbles need to be avoided when working with cells. I am slowly learning tricks to avoid bubbles, since I have been using multiple different kinds of pipettes, and any pipette use can easily lead to bubbles. The graduate student has been sharing different ways to avoid getting air into the various kinds of pipettes I have been using, and I have also been trying to utilize the techniques in new situations I am encountering as well. Also, because of how easy it is to create bubbles when using pipettes, I am open to methods that can achieve the same goal without needing a pipette at all. As I get more practice with pipetting and cells I think I will create less bubbles, but right now I am convinced I will have nightmares about them.

Figure 1. Microscope images of HEK 293 cells from a company that sells them (TGR).

The types of cells I have been working with grow very quickly since they are embryonic, so their media needs to be changed every two days or so and they need split quite frequently. Usually the cells should be split around 90% confluence, which is when the cells are attached to and filling about 90% of the bottom of the dish they are in. If the cells are left to grow, the ones I am working with will start to grow on top of each other.

The analyses I will be using the cells for include protein, RNA, and DAPI staining. I have not analyzed any of the cell samples yet, but I have completed and imaged the Western Blot for the protein and looked at the DAPI staining under a fluorescence microscope to make sure it took. The RNA sample has been frozen down for later, I am not yet sure what analysis technique we will use. The same cell type have and will be frozen down untreated for future use, as they are passage 7 now, meaning that they have been split that many times and all of that time and work should not be thrown away.

I had heard of Western Blotting before I started my time in this lab, but I was not entirely sure of how it was done or the theory behind it. I was able to work on my first Western Blot at the end of this week, and I was sure to read about the process in general and some of the theory ahead of time so I would be prepared. Western Blotting is a technique to analyze proteins in specific samples. Some parts of the Western Blotting process felt familiar because one of the major steps requires an electrophoresis gel that allows the proteins to separate by size. However, because proteins take up different structures, they must be kept denatured to reflect their true sizes. Western Blotting takes a much longer time then PCR/gels - 2 days compared to around 3 hours - because after the electrophoresis, the proteins must be transferred to a membrane and treated with primary and secondary antibodies. The primary antibodies search for and attach to the targeted proteins themselves, and the secondary antibodies attach to the primary antibodies. The secondary antibodies are the ones that are imaged through either fluorescence or chemiluminescence, depending on the equipment available. PCR/gels use dyes that can be imaged through UV lights, allowing direct imaging.

Figure 2. My representation of a Western Blot image when using fluorescence. 

The DAPI staining was a technique I had a natural ability for as I was able to make the slides efficiently and in a way that protected the cells from damage. For the DAPI staining, the cells were grown with a cover slip in the dish - once enough cells had grown onto the cover slip, a slide could be made with the cover slip on top and the DAPI stain between. The funniest part to me about this process is that the cover slips were held in place on the glass slides by simply clear nail polish. Considering how expensive items for the lab can be, it is nice to see that $1 a bottle nail polish does the trick.

Figure 3. The clear nail polish we used to fix our cover slips (Wet n' Wild).

On Wednesday, I went to eat breakfast in the common area and when I was done, I headed over to the lab to start for the day. I was turning the corner and could see the graduate and undergraduate students in the lab but before I could get to them I was stopped by a woman who works in the lab next to us. She told me - as she had told others already - that there was feces on the floor, which some of the other people on our lab floor had already begun to quarantine and get rid of. I went into our lab space to start for the day and found out that the feces was right in the way of the samples we needed to test, and all we could do was wait. The weirdest part was that no one knew how the feces got there, especially since the animals are held in another facility. I don't know if someone did know how it got there but wasn't there or didn't want to fess up, but no matter what, it was quite an odd and unexpected lab hazard.

References:

[TGR]. n.d. HEK 293 cells. <http://tgrbio.com/cellular-models/hek-293-cells/>.

Wet n' Wild. n.d. Wild Shine Nail Color Protector. <http://www.wetnwildbeauty.com/nails/nail-care/wild-shine-nail-color-protector.html>.