Experiment Shadowing

As I was thinking about shadowing somebody’s experiment, I thought shadowing Brennan’s experiment would be a very good idea whose ultimate goal in the project is to isolate RNA from zebra fish’s embryonic stage. As many proteins formation occur during embryonic stage, RNA plays a very vital role, and thus can be isolated from its embryonic stage. He is actually dealing with m-RNA in his project. The goal of his project is also to tag the embryo with GFP (Green Protein Fluorescence) proteins and to see if the embryos fluoresce under fluorescence microscope.

One very important similarity that I found between my project and Brennan’s project was Brennan’s project uses a technique to isolate RNA, which I was also supposed to use in my project to isolate the plasmid DNA from VPS18 protein cells but as we already had our VPS18 DNA plasmid from last year we decided not to isolate them. So, I had no knowledge about running PCR and DNA gel but shadowing his experiment for half an hour gave me a very good idea about those techniques we apply for DNA/RNA isolation. When I shadowed Brennan, he was performing one of the very important sections of his experiment which was to examine if cDNA is viable or not. He was trying to do this by using different primers. I found shadowing Brennan’s experiment pretty productive as I saw how to run PCR and make DNA gel and observe it under UV rays. He explained to me that PCR actually stands for Polymerase Chain Reaction. As DNAs is very small, it’s very difficult to see them so the main objective of using PCR in his project is to amplify DNA. To run PCR, we first need to check if our cDNA is good or not and to check it we run control and experimental samples. If it shows up in control, then our c-DNA is good but if it doesn’t, then cDNA is not good enough, then we can’t tell if gene will be expressed by the data or not but if it shows up in control as well as in experimental, then the gene is expressed. They used cDNA instead of RNA because RNA is very unstable and can be easily cleaved by RNAses, thus they use DNA which is directly synthesized from an RNA template (Peterson, Freeman). It was also interesting to watch him setting up the solution for PCR. I have not used the chemical DNTP in my project but this chemical is very important for his project (it could have been important in my project too if I had isolated DNA from VPS18) as they are the building blocks for new gene replications. I also learned how buffer (PAQ5000) increases efficiency and enzyme (PAQ5000) makes the copy of nucleotides. Learning to make a PCR solution was a totally new technique for me.

Zebra fish embryo with GFP

Yeast cells with GFP

The other similarity that I found between Brennan’s project and mine is both of our projects ultimately deal with GFP cells but the only difference is he is trying to tag them in a zebra fish embryo but I am trying to observe them in yeast cells. We both will use fluorescence microscope in order to look at those GFP proteins tagged in embryo (his project) or in cells (my project).

Even though there are some similarities between Brennan’s and my project, I found most of the things between our projects are different including lab techniques we apply, our primary objective, and other sub-goals etc. The difference is obvious as I am doing my research on yeast which is a primitive fungus whereas he is working on a zebra fish which is a vertebrate.  It’s true that both of our researches deal with proteins but unlike Brennan who is focusing on production of protein for the development of an organism, I am dealing with the life cycle of protein in cells. His project observes the proteins through mRNA but my project observes protein (GFP) by its interaction with other proteins (VPS18). Brennan’s project tries to find the answers for questions like where the RNAs are and how the protein formation occurs in mRNA but my project searches answers for questions like how the specific protein (eg GFP) are made, where do they go when yeast grow etc. His project basically deals about nucleic acid isolation whereas mine deals with change in proteins in hybrid yeast cells due to mating. He is dealing with the proteins in embryonic stage whereas my project deals with the matured yeast cells.

Brennan also gave me a brief idea about making DNA gel and how that is important in his experiment. He showed me how Agar, TAE and ethylene bromide (interpolating agent) forms a gel. He explained to me the fact that DNA is negatively charged and move to the positive charge and how the smaller fragments move faster. Observing the DNA ladder formed from that process was pretty interesting. I also came to learn about the fact that knowing the sequence of a vector and gene lets us to estimate the length of the product as well as its purity.

Overall, I would say shadowing Brennan’s experiment was really fruitful as I got an opportunity to know about many techniques that I have not used or may be will not be using during this J-term class. But now, if someone comes and ask me about what PCR is, what it does, what DNA ladder does etc, then I will at least be able to give them a superficial idea about what they actually are.

 Peterson, S. M., Freeman, J. L., RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis http://www.jove.com/details.stp?id=1470 doi: 10.3791/1470. J Vis Exp. 30 (2009).

http://www.zeiss.com/c12567be0045acf1/Contents-Frame/434348ca09f28863c1257421004d1819

http://ucsdnews.ucsd.edu/thisweek/2005/nov/11_07_stemcell.asp