Mimulus Final Lab

We conducted a double digest restriction association DNA study on mimulus guttatus. The first step was to collect samples which we did on 2 field trips that you can read about https://usfblogs.usfca.edu/eovet/2019/09/17/mount-tamalpais-trip/ and https://usfblogs.usfca.edu/eovet/2019/10/01/second-outdoor-trip/. Next, we extracted DNA from the samples we collected as well as samples previously collected by Alec, the details can be obtained here https://usfblogs.usfca.edu/eovet/2019/10/28/mimulus-leaf-sam…s-dna-extraction/. Next, we double digested our DNA using 2 restriction enzymes; https://usfblogs.usfca.edu/eovet/2019/11/19/double-digestion…adapter-ligation/. Next, we ligated unique DNA barcodes onto each of our individuals, then we used PCR for 2 purposes; to add a second barcode and to test if our library construction was successful; https://usfblogs.usfca.edu/eovet/2019/11/24/dd-radseq-test-pcrfinal-pcr/. Our PCR was successful. After the test PCR, we did a larger reaction that would be used for the following steps; This was the last step that we were able to do as a class. In a perfect world, we would do the following steps; the first would be size selection. Size selection selects DNA of specific sizes, specifically, we would target 400 to 600 bps. Size selection can be done by an automated system called PippenPrep of which we have one in Suni Lab. A second way was to do gel extraction and the final way was to use magnetic beads to isolate the DNA. After size selection, we would then normalize our DNA samples, this means to bring all our DNA samples to approximately equal concentrations. The final step would be to combine all of our size selected normalized PCR products into one vessel. And then, we would run these samples on any illumina sequencer, ie. Wall-e. Sequencing would take approximately 6 hours and if successful, would generate tens of thousands of reads. These data would be run through a bioinformatics pipeline by Zimmerman’s Lab. Ultimately, we would align these data with the published Mimulus data and call SNPs. Finally, we would use these SNPs to infer population differentiation using FST and assess population genetic diversity looking at things like allelic diversity. Based on my knowledge, I would expect to have high FST values which would suggest high differentiation between Mimulus guttatus populations.

DD-RADseq (Test PCR+Final PCR)

On Tuesday, 11/19/19, we started our lab session. Our first task was to conduct a test PCR to test for successful library construction our Mimulus guttatus samples. We first labeled 8 PCR tubes from 9 to 16. Then, we prepared 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 pure water. After that, I added 1uL of each library DNA template to their corresponding tubes along with 15uL of master mix and ran PCR using “PCR1” on BIORAD #1/2. Next, I ran the products of PCR1 for each sample on a 1.5% agarose gel with a 100bp ladder at 130V for 40 minutes. We moved to the second phase of the lab as we completed the test PCR. For the next PCR run, we had to add the special second barcode sequences and the illumina primers to our libraries of Mimulus guttatus, allowing us to identify which specific individuals a given sequence comes from. First, I labeled 8 tubes from 9 to 16, then, I 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. After that, I added 3uL of each template DNA to their corresponding tubes along with 22uL of master mix. I vortexed and spinned down the tubes in microcentrifuge and ran “PCR2” on BIORAD #2. Finally, I ran 2uL of the products of PCR2 on a 1% agarose gel with a 100bp ladder.

Double Digestion and Adapter Ligation

On Tuesday, 11/12/19, we 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. Then, I added 6uL of each of my samples’ DNA in the corresponding tube. We prepared 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. I added 3uL of master mix to each sample and sealed, vortexed, centrifuged and incubated the tubes at 37 degrees celsius for 8 hours. After that, we started our second task, adapter ligation for RADseq. I added 1uL of the working stock EcoRI adapters , EcoRI_8 and EcoRI_9, to my samples with sample IDs 15 and 16. We 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, I added 3uL of master mix to the digested DNA and sealed, vortexed, centrifuged and incubated the tubes at 16 degrees celsius for 6 hours.

PCR Reactions

On Tuesday 11/05/19 at 2:30 PM, we started our lab session which involved PCR reactions. 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; D4, D5 and D6 and added 19 ul of master mix along with 1uL template from EO cATB and JP MW 03 and SHOR 004 to each tube respectively. Finally, I vortexed the tubes and stored them in ice.

Lab 9 – Gel Electrophoresis

On Tuesday, 10/29/19 at 02:20 pm, we started to undergo gel electrophoresis of our mimulus DNA samples from last week. First, I dotted out 16 loading dye dots on a sheet of parafilm. Then, I pipetted 3 uL of each of my PCR products into its own dot and loaded all dots into the gel. Finally, I ran the gel at 130 volts for 30 minutes.

Mimulus Leaf Samples DNA extraction

On 10/22/19 at 02:30 PM, we started to undergo Modified Alexander et al. tube protocol for DNA extraction. First of all, I labeled 3 2.0 mL tubes with the sample codes as follows; EO01, EO02, AND EO03. Then, I added three sterile 3.2-mm stainless steel beads to each tube. I added a small amount of leaf tissue to each tube; from E.O. cATB to EOO1, JP MW 03 to EO02 and SHOR 004 to EO03, using tweezers and I sterilized the tool after each trial. Next, I loaded the tubes within a tube rack into the modified reciprocating saw rack and Hannah mounted the rack to the saw. I briefly spin down the tubes in the centrifuge to pull plant dust down to the lids. I added 434uL preheated grind buffer to each tube. I incubated buffered grindate at 65°C for 10 min in water bath, mixing the tubes by inversion every 3 min. After that, I added 130 uL 3M pH 4.7 potassium acetate, inverted tubes several times and incubated on ice for 5 mins. I span the tubes in a centrifuge at maximum force for 20 mins. I labeled new 1.5 mL tubes with the sample IDs as follows; EO-01, EO-02, EO-03. I transferred the supernatant to these sterile 1.5mL micro centrifuge tubes. I added 600 uL of binding buffer to each tube. Then, I applied 640uL of mixture from the previous step to Epoch spin column tubes and centrifuged for 10 min. After that, I disposed the buffer into the hazardous-waste container. I repeated this step with the remaining volume. I washed the DNA bound to the silica membrane by adding 500uL of 70% EtOH to the column and centrifuge at 15,000 rpm until all liquid has passed to the collection tube and discarded the flow-through. I repeated the previous step and discarded the flow-through again. After that, I centrifuged the columns at 15,000 rpm for an additional 5 min to remove any residual ethanol. I discarded the collection tubes and placed the columns in sterile 1.5 mL micro centrifuge tubes with the labels as follows; EO cATB 10/22/19, JP MW 03 10/22/19, SHOR 004 10/22/19. I added 100uL preheated pure sterile H20 to each tube and let it stand for 5 min. Finally, I centrifuged the tubes for 2 min at at 15,000 rpm to elute the DNA.

Phylogenetic Inference

On Tuesday, 10/08/2019 at 02:30 PM, we’ve started our lab session. We had to use to edit the alignments we made last week, but as my Geneious free trial has expired, I couldn’t work on my own project, instead, I had to work with Jason. We first cleaned up his alignment by editing it down. Then, we used a program called JModelTest2 to figure out the best model of molecular evolution for his sequences. We downloaded the jModelTest2 from canvas and then we went back to Geneious and exported his alignment in Phylip format. We opened the exported alignment on JModelTest2 and computed likelihood scores. After the calculations were done, we chose the best model based on Akaike Information Criterion and Bayesian Information Criterion. In Geneious, we looked at the choices for a MrBayes analysis. After that, we used maximum likelihood to infer a phylogenetic tree of his aligned data set.

An Introduction to Geneious

On Tuesday, 10/01/2019 at 02:00 PM, we started out dry lab session using Geneious software. I’ve previously downloaded this software during summer to analyze sequences from Prof. Zimmerman’s Lab, so my free trial expired prior to lab session. I had to connect to the operating system on the basement server that is hosting the 2 Geneious floating licenses. Next, I selected the local folder and created a new folder called “Molecular Ecology Fish Barcodes”. After that,  I downloaded the classes DNA barcoding sequences from Canvas. When the folders were downloaded, I dragged and dropped them into my Fish Barcode Folder. I created another file called “Emre Ovet Fish Barcodes and dropped two forward and reverse sequence files there. Then, I assembled the forward and reverse sequence reads of the same sample. I opened those assembly sequences, trimmed the low quality bases and fixed the ambiguities. Next, I generated consensus sequences and blasted them. The top hit for my sequence EO01 was Pseudomonas fragi which is a gram negative bacterium and the second hit was Clarias gabonensis which turns out to be a catfish species. This sample was labeled as Coho Salmon in the supermarket. The top hit for my sequence EO04 was Thunnus albacares which is Ahi Tuna. It was labeled as such in the supermarket. After the Blast results, I’ve builded alignments with my sequences. EO01 didn’t match the species I was supposed to have been served but EO04 did. Moveover, EO01 had a total of 106 polymorphic sites and EO04 had 40.

Second Outdoor Trip

On Tuesday 10/25/2019 at 02:00 PM, we took a ride with Alec to a location nearby Mt. Tamalpais (37°53’22.0″N, 122°37’46.7″W). Here, we observed a perennial mimulus population next to a fresh water source. We talked about the possible benefits of this wet habitat for the survival and reproduction of the species and we analyzed an opened mimulus flower.

After filling our bottles with fresh spring water, we took another short car ride to a different location (37°52’37.5″N, 122°34’52.2″ W). We walked along a creek that brought us to a shady environment that was surrounded by trees. Our task was to find another mimulus population, but the plants in this area didn’t blossom at the time which made the task more difficult.

While looking for the mimulus, we discussed that the mimulus population in this environment can survive only for a few months because floods take place in the area during winter. After someone found a mimulus, Alec was able to collect leaf samples from the plant, thus, our goals for this trip were accomplished and we made our way back to campus.

Lab 4

On Tuesday, 09/17/2019 at 02:15 PM, we filled the gel electrophoresis lane table for each of our samples, I decided to place my samples with ID numbers EO 1, EO2, EO3 and EO4 to 5th, 6th 7th and 8th lanes respectively from left to right. Then, we started to undergo the procedure for the Electrophoresis of PCR products. I got my PCR tubes and thawed them.  I pipetted 3ul of my PCR products into its own dye dot on a sheet of parafilm. I loaded 5ul of my dots into the gel. We ran the gel at 130 volts for 30 minutes. After that, we moved to the clean-up procedure of PCR products for sequencing known as ExoSap. I carefully labeled new 0.2 ul PCR tubes with each of my sample codes. We determined the number of PCR clean-ups as 18. We calculated that we needed 191 ul of H20, 22.5 ul of 10x buffer, 7.9 ul of SAP and 4 ul of Exo. I put my reagents on ice and pipetted 7.5 ul of each of my PCR products into a clean, labeled 0.2 ul PCR tube. We made the ExoSap master mix and kept the reagents on ice while it is made. I pipetted 12.5 ul into each PCR product tube and placed my tubes in a thermocycler and then Prof. Paul started the EXOSAP program.