Following up from the prior blog post, this research project requires a lot of trial and error, which has proven to be very tedious and requires a lot of patience. Since the last blog post, we have yet to successfully extract the RNA required to perform the gene insertion into. This could be due to the fact that the primers that have been designed are not binding to the specified binding sites and cutting the RNA enough for it to be visible in the gel.
As previously mentioned, there have been a lot of PCR conditions that were previously tested and manipulated but we have yet to find the right one for the experiment that will give us the results we need. Other changes we have made to the experiment were creating dilutions of the RNA because it actually found in a capsid – or shell, that may be hard to break if using too much concentration of RNA. When using the dilution of the RNA, there is less to work with, therefore may be easier to break the capsid. Once the dilutions were made – 5x,10x, 20x; a gel was run but the results we hoped to obtain did not work.
The two main research goals for the rest of the summer and the rest of the research project is to extract RNA by creating the right primers and conditions, and successfully inserting the GFP gene into the RNA of the parasite.
From this research, I have found that the patience is definitely key because we aren’t just tweaking one part of the research for the results we require, but are actually tweaking multiple aspects of it at the same time and seeing what works and where. Also, this research helped me to be a lot more focused on minor details because it could be the smallest thing that needs to be adjusted or changed to create a very major difference.
Thankfully, I believe that my professor and this research project has prepared me for the steps I will be taking once I graduate in the spring. This research has helped to perfect my skills in various techniques such as PCR and proper pipetting that will be useful when I work in a lab as a forensic scientist. Hopefully, the results in this research will positively impact the science community and further studies and research will be conducted to expand on this topic.