We conducted a double digest restriction associated DNA study on mimulus guttatus.The first step was collecting sample which was done on two field trip which can be read about here. Next, we extracted DNA from the sampled we collected as well as previously collected samples from our boy, Alec. Then we double digested out DNA using two restriction enzymes, which can be read on here. These enzymes cut up the genome into many pieces. Next, we ligated unique DNA barcodes into each of our individuals (the procedure being here). Our next step was to use PCR to add a second unique barcode and to test if our library construction was successful (procedure here). Our PCR was successful as evidenced by the gels that were not photographed. After the test PCR, we did a larger reaction (25 microliters) that was identical that we did with the next step. This was the last step we did as a class.
In a perfect world we would have done the following steps. We would do size selection in which we select DNA of specific sizes and we would target 400-600 bps. Size selection can be done in 3 ways, with pippen prep (automated system) in the Suni Lab #Suni, the second way with gel extraction, or we can use magnetic beads to isolate the DNA fragments as well. After size selection, we would then normalize our DNA samples, this means to bring all of our DNA samples to approx. the same concentration (making more likely the same number of DNA fragments to be sequenced). Final step would be to combine all size selected PCR products into one vessel (or 2). Next, we would run the samples on an Ilumina sequencer (out Iseq1000/walle). Sequencing would take approximately 16 hours and if successful, would generate 10s of millions of reads. These data would be run through a bioinformatics pipeline (help Dr. Z). We would align these sequenced data with the published mimulus guttatus genome and call SNPs. Finally, we would use the SNPs to infer population differentiation using a metric like Fst and assess population genetic diversity by looking at number of alleles, allelic diversity, etc. Based on what I know about mimulus guttatus, I expect populations that are geographically divergent to be genetically divergent. #MolecularEcologyForever