PCR Troubleshooting

24 February 2023

By: Brendan Bauman and Rebecca Fasching

Our group pipetting our next gel

            This semester we are studying the brain and behavior of Green Anole lizards (Anolis Carolinensis). Our group is focusing specifically on studying the Stabilin 2 gene, otherwise known as STAB 2. We are currently researching the function and pathways of our gene in order to determine how it interacts with the breeding cycle of the green anole. We are currently in the process of primer troubleshooting three primers we made to amplify the STAB 2 gene.

In the last few weeks, we have worked on our PCR and Gel Electrophoresis on three different Primers for our STAB2 gene. We began last week with creating our agarose gel for our gel electrophoresis of our three STAB2 primers and one beta actin primer each of which contained one negative control (H2O), and one positive control (cDNA of a Green Anole lizard), while we waited, we identified what our gene does, its function, and the pathways it is involved in. Once our gel was set, we ran a gel electrophoresis on the primers, and captured our results by imaging the gel. Once that was done, we consulted our professor as to what our next steps would be. She then determined that changing the temperature gradient of the PCR reactions would help us determine a more appropriate annealing temperature for our primers.

The gel results from our second PCR troubleshooting attempt

            Our initial troubleshooting attempts came back with some primer-dimers but otherwise looked promising. While researching our gene of choice STAB 2, we discovered that STAB 2 codes for a transmembrane receptor protein that aids in creating new blood vessels, a process otherwise known as angiogenesis. We also found that STAB 2 can be found in the vesicle-mediated transport pathway of the body. These results will be used to research further into how the breeding cycle of the Green Anole lizard is affected by STAB 2.

Our work in lab went well and is going well. Our results may have a few flaws, such as primer dimers in our bands and contaminated water, however most of those flaws cannot be controlled by us. As a group, we have gotten better at staying busy and being time efficient. While also continuing to perfect our pipetting, calculating, running a PCR, and running a gel electrophoresis in lab. We still lack, in certain instances, a good way to split up work between us and create a sort of system. Through the lab we have learned how to run a PCR, and gel electrophoresis, along with determining how to find the function, proper primers and correlated pathways of a gene in the Green Anole Lizard, while also finding ways to troubleshoot primers. Overall, we are continuing to expand our knowledge on the STAB 2 gene in Green Anole Lizards while perfecting what we already know.

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