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.
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.
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.
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.
On Tuesday 09/10/2019, we took a car ride to Mount Tamalpais Rock Spring (37,9106721, -122,6129603). This public land contains 40 endemic plants and it is administered by different entities. The main goal of this trip was to get the full spectrum of what ecologists does. We walked down through rock spring and followed a creek to find and collect samples of Mimulus guttatus.
On our way, we encountered a poison oak. We had to avoid any contact with this plant because it causes irritation on skin.
We stopped at a shady spot where we saw groups of Mimulus guttatus growing. Alec, a graduate student doing research in this area, gave us a brief background on his research. He told us that he is studying time and variability hypothesis by comparing inland and coastal populations of Mimulus guttatus and that the shady part of the land is a good place to study this topic because there is not much temperature fluctuations. He also added that this species has a lot of morphological diversity which made allowed them to survive in different habitats. Moreover, these properties of Mimulus guttatus allowed it to be the second most studied plant species in ecology.
After the briefing, we collected fresh plant leave samples, placed them in tubes with silica gel and labeled them by date and our initials.
After that, we moved to a drier environment and found a dead population of Mimulus guttatus with a different morphology than the ones inside the creek.
Next, we took short ride to a serpentine habitat which is a harsh place to live for plants due to heavy metals inside the soil. We were able to find another dead population of the species.
Finally, we walked through a hillside where we observed a blossomed Mimulus guttatus. Here, we argued that the habitat influences whether the plant are annual or perennial.
On Monday, 09/02/2019 at 18:37, I’ve bought a frozen Coho Salmon fillet and a Wild Ahi Tuna steak from Lucky Supermarket at 1750 Fulton Street. Later, at 19:03, I’ve a bought Pacific Yellowtail sashimi and Long Fin Tuna sashimi from SQwers Japanese Restaurant. I’ve kept the samples at the fridge over the night.
On Tuesday, at 14:00, I’ve started the lab work of extracting DNA from animal tissues. First, I recorded the information about my samples on the Animal Tissue DNA Extraction data sheet. I gave my Coho Salmon sample the ID code “EO-1”, Pacific Yellowtail sample “EO-2”, Long Fin Tuna “EO-3” and Ahi Tuna “EO-4”. Then, I’ve put on gloves and labeled 4 locking rid micro centrifuge tubes both on the top and side for each of my samples with the unique ID codes. I took my samples to free table to cut out a small piece out of them using a scalpel and a tweezer. I wiped my equipment with ethanol after handling each sample. I weighted all my samples and they were in the range of 2-10 mg. I added l00 micro liters of Extraction Solution to each of my labeled sample tubes using a p200 micro liter micropipette and unfiltered tips. Later, I added 25 micro liters of Tissue Preparation Solution to the micro centrifuge tubes with the 100 micro liter of Extraction Solution and I pipetted up and down gently to mix using a p200 micro liter micropipette and unfiltered tips. Next, I added each sample to its corresponding extraction micro centrifuge tube using forceps. I took a disposable non-filtered pipette tip and used it to gently mash my tissue samples up a bit. For each sample, I used a different tip. After that, I incubated the sample at room temperature for 10 minutes. During the incubation, I put the excessive fish samples back into the tubes and placed them on Prof. Paul’s desk. I moved my samples to the heat block. Incubate the sample at 95C for 3 minutes. At the end of 3 minutes, I took the sample out of the heat block, added 100 micro liters of Neutralizing Solution by using a p200 micro liter micropipette and filtered tips and mixed by vortexing. I changed the tip after each pipetting. Finally, I put my samples on ice.
I’ve started the second phase of the lab work, that is the amplification of CO1 from fish. First of all, I’ve labeled 4 centrifuge tubes with 1:10, name of my samples and my initials, both on the top and on the sides of the tube. Then, I added 18 micro liters of purified water to each tube. I added 2 micro liters of my gDNA samples to their corresponding tubes. After that, I gently flicked the tubes with my finger to mix the solutions. I let my lab partners to prepare the master mix using 102.4 micro liters of water, 160 micro liters of REDExtract-N-Amp PCR rx mix, 12.8 micro liters of forward primers and 12.8 micro liters of reverse primers. Next, I wrote the labels of my gDNA samples on my PCR tubes on the top and on the side just below the lid. I added 2 micro liters of 1:10 dilution of my gDNA to each of my PCR tubes. I changed tips between samples. While I was pipetting 18 micro liters of the master mix into my PCR tubes, I noticed that the master mix was almost empty, which points out that either there was a miscalculation of the amount we needed or some of my lab partners pipetted excessive amounts of master mix into their tubes. I obtained a clear master mix from another table and added 18 micro liters into my remaining tubes, that are, EO-3 and EO-4. I changed tips between samples. Finally, I left my PCR tubes on ice next to the thermocycler and then we placed the tubes in the thermocycler and started the reaction.
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