Some Success

Here is one example of the work I have been doing over the past few weeks:

This past week I got the result from my western Blot. After the difficulties with the first bisulfite conversion, it was a nice change to see some expected results. In this western blot we used MCF7 cells and probed using primary antibodies for Tubulin, Ecad, B4, and Actin. The secondary antibodies were Rabbit, Rat, and Mouse. These antibodies bind to the sites of the proteins. After developing the results one can see/compare the amount of proteins in each phase of the cell. The two groups of cells we looked at were Hypoxia and Normoxia. Normoxia is the characterization of cells that have grown in normal oxygen levels. Hypoxia is the characterization of cells that have grown in an atmosphere of 0.5% oxygen. This is achieved by using a machine that keeps the conditions at 0.5% oxygen. In our results, we saw the Ecad decreased from normoxia to Hypoxia, and Beta4 also decreased from normoxia to Hypoxia.

To give an idea of what a western blot entails this is the protocol I had to follow:

1. Make the Acrylamide gel
-two separate gels-stacking gel and Resolving gel-the stacking gel is where you load the samples and the resolving gel is where the samples pass through when an electric current is added.

2. Prepare the samples- Acrylamide gels are for proteins. After extracting a lysate from the cells, you measure the concentration using a bradford machine. Using those concentrations you prepare each sample with equal volume of proteins. If the concentrations differ, the volume will differ. In this case you add Ripa buffer to the lesser volume to make them equal.

3. Add 6x buffer to each sample

4. Load/run the gel. Load gel with Markers as well.

5. With an 8% gel it usually takes about 20-25 Minutes to run.


After the gel runs you transfer the samples on the gel to a membrane using a transfer aparatus. You run this in a cold room at 350 Amps for 90 Minutes.

This is only the first half of a long process. Later in the experiment you have to probe for the proteins and develop the results in a dark room.

I have had success with the western blots and am doing another one today.



Overall the past few days have been really busy. I have been running a lot of experiments, and am learning a tremendous amount. Next week, I will be giving my presentation to the lab on the data I have been getting.

Troubleshooting

Over the past two/three weeks I have been busy with the bisulfite conversion. I used a Qiagen kit and its protocol to perform the procedure. However, although the protocol is straight forward, I have yet to have any success. My first conversion consisted of extracting the DNA, converting it using the kit, and amplifying the DNA using PCR. However, throughout one of these processes something went wrong. So.... Bryan and I started troubleshooting:

-The PCR Cycles- we tried different time cycles to see if that has an effect on the primer binding to the DNA

-The original Genomic DNA- we ran this through an agarose gel to make sure we had DNA to begin with

-The annealing Temps.- we ran the bisulfite converted DNA through a PCR machine with a gradient. We could then see the optimal annealing temperature.

-My technique- we will soon do the procedure side by side.

Despite testing for all of these issues, every time we ran the converted/amplified DNA through a gel we did not see anything in the gel, except for Primer Dimer, which tells us the the primer is not binding to the DNA. I attempted this conversion six times and ended with no results. I was frustrated, but Bryan told me, "thats Science- lots of Troubleshooting." Thankfully, I have moved on from that conversion. Eventually Bryan and I will do it side by side, to test for any human error.

Now I am doing a western blot and will update the blog as soon as I get the results.

Lab Techniques

Over the past three weeks Brian has been showing/teaching me the lab techniques I will need to know for my project. My first few attempts, even with the guidance of Brian, were somewhat failures. Aside from learning the steps, my data did not match what was expected. The very first experiment I ran was extracting the RNA. RNA is extremely fragile and not as stable as double stranded DNA. Because of this I had to use (RNA-Free) lab tools. My pipettes needed to be clean and safe for the RNA to remain intact. However over the course of extracting the RNA, running it in a PCR and loading it into an agarose gel, I must have messed something up, because when we went to take a picture of the gel, there was no result at all. Brian admitted that for my first time doing a PCR, the amount of samples may have been too much. So, the next day, I ran another one with only a few samples, and finally we saw some results-results that Brian had already gotten in his previous lab work. Science can be very tedious. One mistake can set you back to the beginning of the experiment. Regardless, I have been learning a lot. I have several techniques down: RNA extraction, PCR with RNA and DNA, electrophoresis with 1.5%agarose gel, Western Blotting, transferring a western blots to a membrane, and developing the membrane in a dark room. Just recently I have been working on a bisulfite conversion. Over the last few weeks, Brian has taken a few steps back and now allows me to complete the work with little assistance. I do not have to ask him as many questions, and he is beginning to trust that I am following the protocols correctly. Overall it has been an exciting/edifying experience, and I look forward to learning much more as the days continue.

I will upload some pictures of the lab and some of my results with explanations soon.

First Few Days

My first few days have been great. I was not exactly sure what I was going to be working on, but Brian has helped me tremendously with the lab techniques and background for my summer research. Initially, I thought I would be working on my own independent project that did not necessarily contribute to the lab's research as a whole. However, upon arriving I was told that it was just as important that the lab benefit from my work. I became acquainted with the other lab members the first day; they too, are extremely helpful. I have found that they are willing to help me with any of my questions regardless of how busy they may be. The lab area is nice, and I have two desks; one for lab work, the other for writing my lab book and reading.

As far as work goes:
I usually arrive at the lab between 8 and 8:30. On monday/wednesday/friday I have a pathology grad course at eight. A lot of the material is over my head, but I have picked up some of the major concepts. It is nice to hear from experienced doctors on a topic that is so critical in life today. The course relates to the work I am doing in the lab nicely. The course is two hours long: a one hour lecture- and a one hour microscope section. After the class ends, I walk to the lab. Brian gives me an overview of the work that day, and I begin. The work day ends whenever I finish my lab work for that day. The hours are flexible, and Brian cares only that I get the lab work completed.