December 6, 2019
We conducted a Restriction Double Digest Restriction DNA study on Mimulus guttatus. The first step was collecting samples which we did on 2 field trips. Details of the protocol to complete these steps can be found here: https://usfblogs.usfca.edu/evangelinab/ . Next, we extracted DNA from the samples we collected as well as sampled previously collected by our golden boy, Alec. Details of the protocol following steps of extraction can be found here: https://usfblogs.usfca.edu/evangelinab/ . Next, we double digested our DNA using two restriction enzymes. These enzymes cut up the genome into many smaller pieces. Details for how the double digest was completed can be found here: https://usfblogs.usfca.edu/evangelinab/ . Next, we ligated unique DNA barcodes onto each of our individuals. Our next step was to use PCR for two purposes which are; 1. to add a second unique barcode and 2. to test if our library construction was successful. Our PCR was successful by evidence of a photo that Professor Paul was supposed to upload but did not because he has better things to do. After the test PCR, we did a larger reaction of 25 micrometers that is identical. this is the last step that we were able to do as a class. Details of the protocol for how the two PCR tests were run can be found here: https://usfblogs.usfca.edu/evangelinab/ . In a perfect world, we would do the following other steps. However, Professor Paul will try to do these steps over the break while I sleep. Hypothetically, the next step would be size selection. Size selection selects DNA of specific sizes. Specifically, we would target approximately 400-600 base pairs which is typically a good size to sequence with Illumina. Size selection can be done in 3 different ways using an automated system called Pippen Prep of which we have housed in the Suni lab #Suni #doc #bees #pollen. A second way is to use gel extraction. Lastly, we could also use magnetic beads to isolate the DNA. After size selection, we would then normalize our DNA samples which means we would bring all of our DNA sample to approximately the same concentration. Having equal concentrations makes more likely equal number of DNA to be sequenced. The final step would be to combine all of our size selected normalized PCR products into one vessel. Then, we would run these samples on any Illumina sequencer. For our class, we would run them on our in house I-seq1000 a.k.a. Wall-e. Sequencing would take approximately 16 hours and if successful, the sequencing would generate tens of millions of reads. These data would be run through a bioinformatics pipeline (sorry Dr. Z). Ultimately, we would align these sequence data with the Mimulus guttatus genome and call SNPs. Finally, we would use these SNPs to infer population differentiation using a metric like FST and assess population genetic diversity looking at things like the number of alleles, allelic diversity, etc.. Based on what I know about Mimulus guttatus, I might expect populations to be geographically divergent.
