15 September 2023
By: Georgia Deml and Rebecca Fasching
This semester has already shown some unique challenges. The first of these challenges came with combining the remnants of our groups from last semester. Combining our groups meant that we would now have two genes to research rather than last semester’s one per group. This means that we had to find a way to balance our workload as each gene had differing amount of progress in its research. Despite the change in circumstances, we have been adapting well and have been making good progress towards our goals.
Continuing with where we left off from our work in the spring, we began performing cDNA synthesis. We were assigned multiple mRNA samples that had been isolated from brain tissues by our cohort in the spring. Using these RNA samples, we were able to synthesize cDNA by mixing calculated amounts of RNA and nuclease-free water along with 2 µl of primer d(T)23 VN to the RNA and water mix. That mix was then denatured at 65℃ for 5 minutes. Then a reaction mix and enzyme mix was added before being incubated at 42℃ for an hour. After incubation the mix was once again denatured, this time at 80℃, to stop the reaction being performed by the enzyme mix. After this second denaturing, our cDNA was ready to be stored for future use.
During our hour of down time while waiting for our cDNA samples to synthesize, we continued working on Georgia’s gene PLD4. Last semester the PLD4 gene was not having PCR cleanups work successfully enough to be able to be sent for sequencing. Therefore, we have run two new PCR reactions with all three of the primer sets and two different cDNA samples. In our first PCR, we used cDNA 45 and came across what appeared to be primer dimers and contamination of the water wells. Although we did have very dark bands in every well, the bands were not long enough to be able to continue with. We ran a second PCR reaction with all three primers once again and a different cDNA sample (cDNA 49). This gel image showed up very faint in 4 out of the 6 wells and not at all in two of the wells. Because our gels have not been successful, we cannot send out PLD4 for sequencing just yet. Our next step is to redesign 3 new primer sets for PLD4 and continue troubleshooting with this gene.
We have made good progress since we started our work for this semester. Our next steps are to redesign primer sets for PLD4 and to test the cDNA samples we have made. We will test the cDNA samples by creating PCR samples using the cDNA we made. Once we have new primer sets for PLD4 we can start troubleshooting those primers. If the new primers are effective we can move forward in researching PLD4 and send in a sample for sequencing.