28 February 2020
By: Alexus Bunnam and Taylor Grossen
Introduction:
The primary purpose of our research in the Brain and Behavior stream is to determine the upregulation of a specific gene between the breeding and nonbreeding season of Anolis carolinensis lizards. Before research began, we had to ensure we properly knew how to use the PCR thermocycler and gel electrophoresis machine, which we did by doing a series of protocols with a control primer set (beta actin) that was given to us.
Research:
Our research began once we decided upon our gene, PER1. PER1 is a circadian clock gene that is located in the superchiasmatic nucleus of the brain. After deciding upon our gene, we had to design three primers sets for our specific gene, which we did by using the National Institutes of Health NCBI and IDT websites.
After receiving our primer sets, we were able to start our research. Over the past couple of weeks, we have been running multiple PCR’s and gels. The making of PCR starts off with creating a master mix, which is composed of Go Taq, a forward and reverse primer and PCR water. Since we had three primers and a control, we had a total of four master mixes. Our results from our first PCR and gel were inconclusive. Based on our gel image multiple things went wrong: the image was extremely fuzzy which means there was something wrong with the gel. Our beta actin band did not show up which is our control variable, meaning there was something wrong with our PCR process. Also, our wells containing water controls had bands in them meaning they were contaminated. We decided for our second PCR we would do the exact same protocol. Upon starting our second PCR, we found out that all the liquid within our forward primer one, was gone. While vortexing we may have melted a tiny hole in the tube causing the liquid to leak out. Due to this, we omitted the primer one set. After performing the same protocol, results showed the second time around that we had made a better gel, but again we saw contamination in our wells that contained water which may be due to pipetting errors. However, on the upside, our wells containing cDNA had no contamination and the bands were extremely dark.
We needed to be sure of our results, so again, we did the exact same protocol and we noticed that the bands on our primers were still extremely dark (third PCR). With these results we decided that our primers were too concentrated and therefore, in our next protocol we will need to perform a dilution.
Conclusion:
We have experienced many failures, but in research we know that this is part of the process. We’ve been frustrated, confused, and discouraged many times. If not with the help of our peer mentors, TA, and professors, we would be completely lost. We have yet to achieve full success in running a PCR. Future plans include troubleshooting PCR, and eventually performing a PCR clean up.
