We constructed a double digest restriction associated DNA study on Mimulus guttatus. The first step was collecting samples, which we did on two field trips which can be read about here and here . Next, we extracted DNA from the samples we collected as well as samples collected by Alec detailed here. Next, we double digested our DNA using two restriction enzymes detailed here. These enzymes cut up the genome into many pieces. Next, we ligated unique DNA barcodes onto each of our individuals. The next step was using PCR for two purposes: to add a second unique and to test if our library construction was successful. Our PCR was successful as evidenced by a photo taken by Professor Paul. After the test PCR, we did a larger reaction of 25 micrometers that is identical to the previous one. This is the last step we were able to do as a class. In a perfect world, we would do the following other steps. The next step would be size selection. Size selection selects DNA of specific sizes, specifically, we would target ~400-600 bp. Size selection can be done in three different ways, one is using an automated system called pippin prep of which we have one housed in the Suni lab #Suni. A second way is to use gel extraction. Or, finally, magnetic beads can be used to isolate DNA. After size selection, we would then normalize our DNA samples, meaning bringing all of our DNA samples to approximately the same concentration. Having equal concentrations makes equal numbers of DNA fragments more likely 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 alumina sequencer- our class would run it on our in-house iSeq 1000 (Wall-e). Sequencing would take approximately 16 hours and, if successful, would generate tens of millions of reads. These data would be run through a bioinformatics pipeline. Ultimately, we would align these sequence data with the published 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 that are genetically divergent. #MolecularEcologyForever
