Blog Post 2: Inflammatory Lipids: Differences in Male and Female Production

So far in our research we have written a procedure for genotyping and successfully genotyped one of the litters. We worked off of a previously written protocol and adjusted things as were necessary, adding in more information about locations of reagents and what adjustments we had to make along the process. We performed PCR and gel electrophoresis, twice before it was successful because the gel did not turn out the first time. The first gel produced bands all at the same band length, which indicated that something went wrong in the process. We knew that the error did not occur in the process of running the gel itself because we did get bands and the DNA ladder turned out as well. We believed there was an issue with our PCR (polymerase-chain reaction) product which resulted in this error. The PCR product could have sat for too long before being used or it was not mixed well enough before use. We repeated the process, making sure to be extra careful with our technique this time, especially with pipetting and mixing the PCR products that we generated. This resulted in a successful gel. We then analyzed the gel to determine the genotypes of each mouse and whether they were wild-type (WT), heterozygous, or homozygous for our gene. We have also started writing up another protocol using a previously published protocol for protein calibration assays which we hope to start in the spring semester.

I didn’t know much about the project when we first started and Dr. Upmacis was very helpful in showing me some of the literature and explaining some of the theory behind our project. We worked together in updating the previously written procedure and she has provided me with supervision when working on the gel. This process has required a lot of communication between the two of us, both to figure out times we are both available to work and to problem solve issues that arise. Throughout our research, I have learned how to perform the process of PCR, which I had never done before. I’ve also learned more about making necessary adjustments when experiments are unsuccessful.

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