Since the last blog post, my research has run into many roadblocks unfortunately. These challenges were to be expected though as scientific research comes with much trial and error. The one success that has come out of my research so far this summer was extracting the RNA from the Cryspovirus itself. This was done by first breaking the oocysts, or shells, of Cryptosporidium parvum, which is where the Cryspovirus is housed. However, when attempting to extract the GFP, green fluorescent protein, from the gel it did not work. This raised many questions as to if there was any GFP present at all. To be sure that I did in fact extract GFP present we used vectors and restriction enzymes to be sure that there was GFP present and that the correct size I was looking for. These results came out negative causing me to think of other problems as to why I could not successfully extract GFP.
Along with the GFP I also ran a gel using cas9, CRISPR-associated protein-9 nuclease, which is an enzyme that will help insert the GFP into the virus RNA so that it can fluoresce. I used vectors and restriction enzymes with cas9 also to see if it was present and the results came out positive. So, cas9 was present but GFP was not. Without the GFP being present there would be no use for the cas9 at this point so I just froze down the rest of the cas9 to keep as a stock solution for when the GFP eventually gets extracted from the gel successfully.
I think the reason why there is no band showing up on the gel for RNA is because the hb, ns, and cpv primers are not cutting the RNA correctly meaning the PCR is not copying the correct sequence of RNA. The next step in my research is to design and order new primers that will be used with the RNA extracted from the Cryspovirus. These primers have been designed specifically to cut the RNA at the exact positions, but of course there is no guarantee they will work until I use them to do a PCR. If the new primers do not work then new ones will have to be made until I get primers that bind to the right positions on the RNA to cut the correct sequence.
Throughout my time conducting this summer research I have come to learn many things. First of all, I have learned to be very meticulous while working in the lab because it is very easy to make small mistakes that can cause your research to be affected. I have also learned that there are not always straight forward answers as to why something went wrong and that it takes a lot of time and thinking to solve an issue. However, even if you think you have found the answer as to why something went wrong you may not always be correct. There is a lot of critical thinking and trial and error that goes into research.