We conducted a double digest restriction-associated DNA study on Mimulus gutattus. First, we collected samples across two field trips, described in the following entries:

https://usfblogs.usfca.edu/jkrastins/2019/09/17/summary-of-experiences-from-9-10-2019-field-trip/

https://usfblogs.usfca.edu/jkrastins/2019/10/01/field-trip-2/

Next, we extracted DNA from samples we collected on these trips, combined with DNA gathered from specimens collected by Alec Chiono, a graduate student in Professor Paul’s lab. This was done with the following protocols:

https://usfblogs.usfca.edu/jkrastins/2019/10/29/lab-8/

https://usfblogs.usfca.edu/jkrastins/2019/11/05/lab-9-jason-krastins/

Second, we double-digested our DNA using two restriction enzymes, using the following protocols

https://usfblogs.usfca.edu/jkrastins/2019/11/12/lab-10-jason-k/

By doing this, the genomic DNA was cut into numerous pieces.

Next, we ligated unique DNA barcodes onto DNA gathered from each individual sample, using the following procedure:

https://usfblogs.usfca.edu/jkrastins/2019/11/19/lab-11/

After which, we did several PCR reactions. The first was to add a unique barcode to the samples, and a second was done to test if the PCR was successful. The PCR was successful, as evidenced by photography of the gel. This procedure is also described in the above post.

 

After the test PCR, we did a larger reaction that was identical that will be used for the following steps, described as follows:

https://usfblogs.usfca.edu/jkrastins/2019/11/26/lab-11-jason-krastins/

This was the last step we were able to do as a class, as the PCR was unfortunately not successful this time.

If we had more time to do reactions we would do size selection. We would select for DNA sequences of specific sizes; specifically, we would target 400-600 bp fragments. We would use an automated system known as Pippin Prep, which is contained in the lab of Sevan Suni, gel extraction, or magnetic beads to isolate the DNA. Next, we would standardize the concentration DNA fragments contained across our samples, to allow for uniform DNA numbers for extraction. After that, we would normalize all of our PCR products into one single vessel. Finally, we would run them on our i-Seq 1000 machine in order to sequence our fragments. Sequencing would take approx. 16 hours, and, if successful, would generate ~10-20 million reads worth of data. These data would be run through a bioinformatics pipeline with the help of Dr. Zimmerman; the analyzed data would then be uploaded to the Mimulus gutattus  online genomic database. Then, we would analyze variance using a metric such as FST values to assess population genetic diversity of the individuals  using metrics such as allelic diversity. I hypothesize that the populations would show significant genetic divergence based on distance between populations and preferred habitat types (mesic vs. serpentine growth habitats)

On November 19th, the Pyrimidudes started our lab session. The first thing we did was a PCR to test for successful library construction our Mimulus guttatus samples. To begin, we labeled 8 PCR tubes from 9 to 16. After, we prepped a master mix for RADseq with 11 reactions using 88uL of NEB One-Taq 2x Master Mix, 4.4uL of forward primer, 4.4 uL of reverse primer and 68.2uL of DNA-free water. After that, we each together added 1uL of each library DNA template to their corresponding tubes along with 15uL of master mix and ran PCR using the “PCR1” on te BIORAD #1/2. Next, we each loaded 2 of the products per person in the group ran the products of PCR1 for each sample on a 1.5% agarose gel with a 100bp ladder at 130V for 40 minutes. Then, we collected the gel. For the next PCR run of the day, we added the second barcode sequence along with the Illumina primers to our libraries of Mimulus guttatus, allowing us to identify which individuals. We yet again labeled 8 tubes from 9 to 16, then, we together prepared a master mix with 11 reactions using 3.40uL of Phusion DNA polymerase, 68.80uL of 5x Phusion HF buffer, 17.20 uL of 10uM PCR 2-5 forward primer, 17.20uL of 10uM PCR 2-5 reverse primer, 6.90 uL of 10mm dNTPs, 10.30 uL of DMSO and 118.30 uL of pure water. For the final step, we added 3uL of each template DNA to their corresponding tubes along with 22uL of master mix solution to each tube. We finally vortexed, then spun down the tubes in microcentrifuge and ran “PCR2” on BIORAD #2. To finish off the procedure, I ran 2uL of the products of PCR2 on a 1% agarose gel with a 100bp ladder, which was taken by Prof. Paul and stored.

On 11/5/19, the Pyrimidudes started our lab session. Our first task was to conduct double-digest restriction associated DNA sequencing. I labeled 2 tubes as P5 and P6, for my samples CHIM 002 and PRBM 005. After this, I added 6uL of each of my samples’ DNA to each tube. Next, we worked together to prepare a master mix with 11 reactions using 9.9uL of CutSmart buffer 10X, 3.1 uL of EcoRI-HF enzyme, 1.3 uL of MSPI enzyme and 18.7 uL of pure water and we placed the master mix on ice. Each of us then added 3uL of master mix to each of the 2 samples we were responsible for and sealed, vortexed, and centrifuged. After that, Prof Paul put the tubes in the incubator at 37 degrees celsius for 8 hours.

 

The next lab period on 11/12/19, we started our second task, which was doing adapter ligation for RADseq. I added 1uL of the working stock EcoRI adapters , EcoRI_8 and EcoRI_9, to my samples with sample IDs 13 and 14. The Pyrimidudes prepared an adapter ligation master mix with 11 reactions using 4.4 uL CutSmart buffer 10X, 14.3uL ATP(10mM), 2.2 uL T4 Ligase, 1.1 uL pure water. Then, we added 3uL of master mix to the digested DNA and sealed, vortexed, and centrifuged the tubes. Prof Paul then incubated the tubes at 16 degrees celsius for 6 hours.

Last Tuesday, my group, the Pyrimidudes, started our lab session which involved doing PCR reactions to amplify the DNA we gathered by running gel electrophoresis on our M. guttatus tissue samples gathered the Tuesday prior. We prepared a master mix using 214uL of ddH2O, 32uL of 10x buffer, 32uL of MgCl2, 16uL of BSA, 3.2uL of dNTPs, 3.2uL of F-primer, 3.2uL of R-primer and 1uL of Taq. Then, I labeled 4 tubes with the following tags; D1, D2 and D3, and added 19 ul of the above master mix along with 1uL template from JK CATB 01, and ALC TROJ02, and ALC SHOR 003 to each tube respectively. Finally, I vortexed the tubes and stored them in ice until Dr. Paul could put them in the PCR machine to amplify the DNA for processing next lab period.

Last Tuesday, the Pyrimidudes ran a gel electrophoresis of our Mimulus guttatus DNA samples that were amplified last week. First, I dotted out 13 loading dye dots on a sheet of parafilm. Then, each member of our group I pipetted 3 uL of each of our individual 3 samples of our PCR products into its own dot, pipettied up and down to mix, and then loaded all dots into the gel. After all members had added their DNA plus the ladder, Dr. Paul ran the gel at 130 volts for 30 minutes, then stored the products in the freezer for future use.

For Lab 8, I was preparing DNA extractions from Mimulus guttatus leaf samples. I had three samples: CATB JK 01, simplified to JK01, SHOR 002 AJC, simplified to JK02, and TROJ 003 AJC, simplified to JK03. Although broadly similar to the prior fish DNA extraction, plant tissue is more dense, so we first had to pulverize it using steel ball bearings for 40 seconds using a modified reciprocating saw to masticate the tissue. Following mastication, the tubes were spun down to concentrate the tissue powder, and had 434 microliters of warm grind buffer added to each tube. The grindate was then incubated for 10 min in a water bath, after which 130 mL of potassium acetate was added. The tubes were then incubated on ice for 5 minutes to precipitate out DNA. Next, the sample was vortexed at 15000 RPM in a microcentrifuge for 20 minutes to separate the supernatant from the solid leaf tissue particles. Next, the supernatant was transferred to new 1.5 mL sterile tubes labelled with the sample IDs ( JK01, JK02, and JK03). After that,  I added 600 microliters of binding buffer to bind the DNA to a flow-through collection column. I then passed the buffer solution containing the DNA through an Epoch spin column for 2 cycles of concentration in the column followed by two washes with 500 mL 70% EtOH, with a final 5 min spin cycle to remove the remaining EtOH. After this, the columns were moved to the new, labeled 1.5 mL microcentrifuge tubes, and had 100 microliters of sterile H20 added to collect the eluted DNA. After this, they were spun for a final centrifuge cycle for 2 minutes for elution, then taken away by Dr. Paul and stored on ice for later processing.

For Lab 7, I practiced using phylogenetic tree analysis methods on 10 of the DNA sequences from my DNA isolation project earlier this semester. First, I ran the Jmodeltest program on my assembled BLASt hits in order to determine the best methods for analysis; the AIC gave HKY as the best molecular model, whereas BIC turned up H80. Next, I made a series of phylogenetic trees using different programs to test them out. First, I made a MrBayes tree using the HKY85 model. This gave back an identity suggestings that Red Drum (Sciaenops ocellatus) was the most likely ID for my fish. Next, I did a Maximum likelihood tests with RAxML Bootstrapping, followed by one with PHYML bootstrapping. Both gave similar results as MrBayes, but with less support of side trees. Both gave weak posterior distributions, likely due to insufficient sample sizes.

I assembled the forward and reverse reads results for the JK-02 and PR-03 gene assemblies after downloading the info off of Geneious. Unfortunately, the read quality was too low to be able to assemble the reverse and forward reads of the JK-02 sequences, both of which had about 20 polymorphic sites each close to the ends. It was an interesting discrepancy; the read quality was low, but polymorphic sites were also low. Despite the low read quality, the DNA was successfully blasted and indicated that the fish was in fact the Red Drum, Sciaenops ocellatus. The DNA samples from PR-03 were much better in quality overall and assembled easily without any polymorphic DNA sites. The species ID was not as certain, however, though it was confined to only very closely related species in the genus Thunnus, namely T. alalalunga and T. orientalis.  I was informed that the samplw was supposed to be tuna, but not which species, so I am unsure of whether or not the marked species was correct for that individual.

On Sept. 24th 2019, me and my Molecular Ecology class took a field trip to the coastal areas in the vicinity of Mt. Tamalpais state park. Due to a navigation error, the SUV I was driving took a wrong turn, so we missed some of the initial field trip time. Eventually however, we met up with the main group at a spring by the side of the highway near Stinson Beach. Here, Dr. Paul introduced us to a perennial form population of Mimulus guttatus in order to show us the morphological forms the species can take when exposed to constant moisture, rather than the deceased annual populations we had seen before. After this, he took us to a creek near the Muir Beach trail to show us another population that was on a different annual cycle than the ones one the serpentine barrens. As they grew on a dry creek bed during the summer season and the creek region was less dry in the summer, this population was summer-annual rather than winter-annual. Thus, by observing and sampling them, we get to see yet another form of Mimulus guttatus expression, and add more to our gene library. I also got to see a lot of native fish and crayfish in the creek, which was interesting. Overall, I got to see more new forms of Mimulus than I had seen before, which helped my better grasp the full variation of the species.

For the Pyrimidudes, Lab 4 was all about separating out and then cleaning up the products of our PCR reactions from the last lab section. In order to set up the gel, the Pyrimidudes planned out which samples would go where; in total, we had 13 samples, plus a ladder and control. In order to prep out PCR, products, I pipetted out 15 1.5 microliter dots of loading dye, while the rest of the Pyrimidudes carefully set up the PCR reactions for adding to the dots. Each of our group added 3 microliters of DNA to each dye dot, with the exception of myself, who added the dye dot and PCR products directly into each lane, where they were mixed in-situ by pipetting up and down. We then run the gel at 130 volts for 30 minutes.

As this was running, we next prepped at PCR cleanup using ExoSAP of some of the remaining samples by creating a master mix using 190.62 μL H2O, 22.5 μL 10x SAP Buffer, 7.9 μL EXP, and 3.96 μL SAP for a total volume of 225 μL. We then added 12.5 μL of this master mix to each of 13 different labeled PCR tubes, to which was added 7.5 μL each of our PCR products. The PCR products were then run for 45 minutes by Dr. Paul using the ExoSAP protocol.