We conducted a double digest restriction associated DNA study on Mimulus guttatus

The first step was collecting samples which we did on two field trips which are described in these entries.

  • https://usfblogs.usfca.edu/orieney/2019/09/16/september-10-mount-tamalpais/
  • https://usfblogs.usfca.edu/orieney/2019/09/29/september-24-mimulus-pt-2/

Next, we extracted DNA from the samples we collected as well as previously collected samples (by Alec J).

  • https://usfblogs.usfca.edu/orieney/2019/10/24/october-22-dna-e…-mimulus-gutatus/
  • Gel electrophoresis : https://usfblogs.usfca.edu/orieney/2019/11/03/october-29-gel-e…oresis-m-gutatus/
  • PCR : https://usfblogs.usfca.edu/orieney/2019/11/12/november-5-pcr-r…-mimulus-gutatus/

Next, we double digested our DNA using two restriction enzymes. These enzymes cut up the genome into many pieces and we ligated unique DNA barcodes onto each of our individuals.

  • https://usfblogs.usfca.edu/orieney/2019/12/03/november-12-ddra…adapter-ligation/

The next step was to use PCR to (1) add a second unique barcode [table based] and to (2) test if our library construction was successful.

  • https://usfblogs.usfca.edu/orieney/2019/12/04/november-19-test…reactions-radseq/

Our PCR was successful (picture documented by Prof. Paul).  We threw this away

After the test PCR, we did a larger reaction (25 mL) that was identical and used for the following steps.

  • This was the last step we were able to do together as a class. In a perfect world we would do the following other steps (Prof. Paul will do these over break)
    • Size selection – select DNA of specific sizes, specifically, we would target approximately 400-600 base pair fragments [good size for Illumina]. Size selection can be done in 3 different ways using an automated system called Pippen Prep of which we have in the Suni J …… #Suni
      • A second way is to use gel extraction (probably will use this method). Use a ladder to cut out base pairs
      • Third way- magnetic beads to isolate DNA fragments
    • After size selection, we would then normalize our DNA samples- bring all our DNA samples to approximately the same concentration. Having equal concentrations àincreased likelihood of equal number of DNA fragments to be ultimately sequenced from individuals
    • Final step is to combine all of the size selected, normalized, PCR products into one vessel
    • Then, we would run these samples on any Illumina sequencer. For our class, we would run it in our in-house Wall-E [iSeq 1000]
    • Sequencing would take approximately 16 hours. If successful, it would generate tens of millions reads. These data would be run through a bioinformatics pipeline
    • Ultimately, we would align these sequence data with the published Mimulus gutatus 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 gutatus, I might expect populations that are genetically divergent

# MolecularEcology4EvR


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