The past few months have been amazing. After all of my time in the lab, I am now confident in my ability as a student researcher to not only execute experiments via following procedures but also reflect and troubleshoot challenges I have faced along the way.
Particularly, at the moment, synthesizing RNA is my toughest challenge. As it is a very specific procedure and is easily contaminated, obtaining RNA from my mutant DNA is my goal. Originally, I thought since it was a new protocol, the mistake was in my pipetting. When pipetting amounts such as .5 μl can be a difficult task. However, after repeating the experiment with the help of my more experienced research team members, and still did not obtain RNA, this hypothesis was ruled out. Next Dr. Buraei and I thought that we were not getting results because perhaps my DNA was contaminated. To check this we ran a “test cut”, which is a process by which we cut the DNA using restriction enzymes. Since we know where the enzymes cut and the construct of the DNA plasmid, we are able to mathematically determine how long each piece of DNA is once cut. Below are the results:
Although it may be difficult to see, the right most column contains 2 white bands. Using the ladder to the left (which provides a ruler by which we can determine the amount of base pairs in DNA) we determined that the two bands coincide with the mathematically expected band sizes. Thus, our mutant DNA does contain the desired sequence, and is not the problem.
Currently, I am conducting experiments including various times and temperatures to troubleshoot my RNA synthesis experiment, hopefully in the next blog I can show you all a picture containing my RNA bands!