Mimulus Final Lab

We conducted a double digest restriction association DNA study on mimulus guttatus. The first step was to collect samples which we did on 2 field trips that you can read about https://usfblogs.usfca.edu/eovet/2019/09/17/mount-tamalpais-trip/ and https://usfblogs.usfca.edu/eovet/2019/10/01/second-outdoor-trip/. Next, we extracted DNA from the samples we collected as well as samples previously collected by Alec, the details can be obtained here https://usfblogs.usfca.edu/eovet/2019/10/28/mimulus-leaf-sam…s-dna-extraction/. Next, we double digested our DNA using 2 restriction enzymes; https://usfblogs.usfca.edu/eovet/2019/11/19/double-digestion…adapter-ligation/. Next, we ligated unique DNA barcodes onto each of our individuals, then we used PCR for 2 purposes; to add a second barcode and to test if our library construction was successful; https://usfblogs.usfca.edu/eovet/2019/11/24/dd-radseq-test-pcrfinal-pcr/. Our PCR was successful. After the test PCR, we did a larger reaction that would be used for the following steps; This was the last step that we were able to do as a class. In a perfect world, we would do the following steps; the first would be size selection. Size selection selects DNA of specific sizes, specifically, we would target 400 to 600 bps. Size selection can be done by an automated system called PippenPrep of which we have one in Suni Lab. A second way was to do gel extraction and the final way was to use magnetic beads to isolate the DNA. After size selection, we would then normalize our DNA samples, this means to bring all our DNA samples to approximately equal concentrations. The final step would be to combine all of our size selected normalized PCR products into one vessel. And then, we would run these samples on any illumina sequencer, ie. Wall-e. Sequencing would take approximately 6 hours and if successful, would generate tens of thousands of reads. These data would be run through a bioinformatics pipeline by Zimmerman’s Lab. Ultimately, we would align these data with the published Mimulus data and call SNPs. Finally, we would use these SNPs to infer population differentiation using FST and assess population genetic diversity looking at things like allelic diversity. Based on my knowledge, I would expect to have high FST values which would suggest high differentiation between Mimulus guttatus populations.

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