Mimulus guttatus Lab Report

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.


Lab 12: DDRadseq

November 19, 2019

Test PCR 1

To begin our lab, we tested for successful library construction of our Mimulus guttatus samples (restriction digest and ligation of barcodes) using a test PCR. We first labeled 8 PCR tubes with the sample ID# 17-24. I then prepared a master mix for RADseq which contained; 88μL of NEB one-taq 2x Master Mix, 4.4μL ofForward Primer (PCR1 X), 4.4μL of Reverse Primer (PCR2 6), 68.2μL of pure H2O, and 1μL of the library DNA template. I added the 1μL of each library DNA template to their corresponding labeled tubes and added the 15μL of Master mix as well. The PCR was run using the “PCR1” on BIORAD #1/2. Next, the products of PCR 1 were ran for each sample on a 1.5% agarose gel with a 100 bp ladder at 130V for 40 minutes.

Test PCR 2

In the second part of our lab, we completed a second PCR run. First, we had to ass the special second barcode sequences and the illumina primers to our Mimulus guttatus libraries. This allowed us to identify which specific individuals a given sequence comes from. I started off by labelling 8 tubes with the sample ID# 17-24. We then moved on to prepare the master mix which contained; 3.4μL of Phusion DNA polymerase, 68.8μL of %x Phusion HF buffer, 17.2μL of 10μM PCR 2-5 forward primer, 17.2μL of 10μM PCR 2-5 reverse primer, 6.9μL of 10mm dNTPs, 10.3μL of DMSO and, 118.30μL pure H2O. After the master mix was made, I added 3μL of each template DNA to its corresponding labeled tubes. In addition, I added 22μL of the master mix. The tubes were capped, vortexed, and microcentrifuged. The PCR was run using the “PCR2” on BIORAD #2. Lastly, 2μL of the products from PCR2  were ran on a 1% agarose gel with a 100bp ladder.