We conducted a double digest restriction-associated DNA study on Mimulus gutattus. First, we collected samples across two field trips, described in the following entries:
https://usfblogs.usfca.edu/jkrastins/2019/09/17/summary-of-experiences-from-9-10-2019-field-trip/
https://usfblogs.usfca.edu/jkrastins/2019/10/01/field-trip-2/
Next, we extracted DNA from samples we collected on these trips, combined with DNA gathered from specimens collected by Alec Chiono, a graduate student in Professor Paul’s lab. This was done with the following protocols:
https://usfblogs.usfca.edu/jkrastins/2019/10/29/lab-8/
https://usfblogs.usfca.edu/jkrastins/2019/11/05/lab-9-jason-krastins/
Second, we double-digested our DNA using two restriction enzymes, using the following protocols
https://usfblogs.usfca.edu/jkrastins/2019/11/12/lab-10-jason-k/
By doing this, the genomic DNA was cut into numerous pieces.
Next, we ligated unique DNA barcodes onto DNA gathered from each individual sample, using the following procedure:
https://usfblogs.usfca.edu/jkrastins/2019/11/19/lab-11/
After which, we did several PCR reactions. The first was to add a unique barcode to the samples, and a second was done to test if the PCR was successful. The PCR was successful, as evidenced by photography of the gel. This procedure is also described in the above post.
After the test PCR, we did a larger reaction that was identical that will be used for the following steps, described as follows:
https://usfblogs.usfca.edu/jkrastins/2019/11/26/lab-11-jason-krastins/
This was the last step we were able to do as a class, as the PCR was unfortunately not successful this time.
If we had more time to do reactions we would do size selection. We would select for DNA sequences of specific sizes; specifically, we would target 400-600 bp fragments. We would use an automated system known as Pippin Prep, which is contained in the lab of Sevan Suni, gel extraction, or magnetic beads to isolate the DNA. Next, we would standardize the concentration DNA fragments contained across our samples, to allow for uniform DNA numbers for extraction. After that, we would normalize all of our PCR products into one single vessel. Finally, we would run them on our i-Seq 1000 machine in order to sequence our fragments. Sequencing would take approx. 16 hours, and, if successful, would generate ~10-20 million reads worth of data. These data would be run through a bioinformatics pipeline with the help of Dr. Zimmerman; the analyzed data would then be uploaded to the Mimulus gutattus online genomic database. Then, we would analyze variance using a metric such as FST values to assess population genetic diversity of the individuals using metrics such as allelic diversity. I hypothesize that the populations would show significant genetic divergence based on distance between populations and preferred habitat types (mesic vs. serpentine growth habitats)