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Lab 6: An Introduction to Geneious

October 1, 2019


We began our lab session by downloading a software called Geneious. Geneious is a commercial program that performs many functions on DNA and protein sequence data. I first created a folder where I was able to download and store Fish DNA barcoding sequences from canvas. The folder contained both the reverse and forward reads and contained the chromatograms of our sequences. However, when I clicked on the forward read of my sequence it looks like I did not have a very successful PCR run at all. This may mean that bacteria or fungi could have contaminated my sample or that my sequencing run was a complete fail. Because of this, I used the sample sequences from other students which were much more successful in their PCR runs. I used the files “ARA01” and “BP01” as my new sequences. After I dropped these files into my “Fish Barcode” folder, I was able to begin assembling the forward and reverse sequences of the sample “BP01”. In this step, I was able to examine my sequences and delete any bases on the two ends that were unreadable or trim off any ambiguities. Once that was completed, I generated a consensus sequence and put it into a new file where it was ready to undergo BLAST. BLAST stands for Basic Local Alignment Search Tool and is used to search the database for highly similar sequences. The top hit for my sequence BP01 was Thunnus obesus which is yellowfin tuna. This is exactly the name of the fish that the student was told when they purchased it. For the sequence ARA03, I found that the top hit was Seriola quinqueradiata which is Yellowtail or Japanese amberjack. Once again, this is exactly the name of the fish that the student was told when they purchased it. There were polymorphisms present in both of these sequences. In the ARA03 sequence, I found 599 polymorphisms which were all SNPs. The first 10 polymorphisms were at sites 63, 68, 70, 89, 92, 95,113,128, 131, and 146. In the BP01 sequence, I found 8 polymorphisms which were also all SNPs. The polymorphisms were at sites 9, 275, 291, 363, 384, 405, and 418.

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Lab 5: Finding Mimulus

September 24, 2019


The first stop on our field trip was Mount Tamalpais. When we first pulled into this area, we saw that there was a natural stream of water coming from the mountain. Here we were able to observe a large costal parenial Mimulus. It was important to note that, we found these mats or patches of Mimulus directly next to the water source. Mimulus loves to keep its feet wet so it is not surprising that we found Mimulus near a spring. In a location like Mount Tamalpais, we were able to see how important the bee population is. Usually, the bees are the ones to pollinate the flowers along this mountain side, including Mimulus, which promotes intrabreeding and interbreeding amongst plant species. We saw that the baby Mimulus did not have much space to “move” or pollinate on its own so, it is vital that bees are able to pollinate around the mountain.

Professor Paul brought it to our attention that the Mimulus population we saw could be a sink population. This means that the population would thrive near a body of water but may get flooded out by rainfall before they are able to flower. Additionally, Alec shared that in drought years, Mimulus may thrive better because they have a longer time to flower and germinate their seeds. This would be beneficial because these Mimulus would be more likely to contribute to the next generation. I was able to learn that Mimulus responds to long day periods but this time of year, there are shorter day periods so, it may not be long enough for these Mimulus to get the proper environmental cues to grow and persist.


We then drove a few miles away from the mountain to a location called Redwood Creek. The creek was well shaded and made for a cool and moist environment with patches of sunlight for Mimulus. Our task in this location was to find a Mimulus population that was growing along this creek. The class as a whole was not able to locate these populations but, we managed to find a few small Mimulus with the help of a few careful eyes. At Redwood creek, it seemed that the populations of Mimulus that we found were much smaller and hidden than the past populations that we had seen. It was explained to us that plant species that live along Redwood creek are susceptible to flooding during the winter time. This, again, is detrimental to Mimulus because they are not able to properly germinate and contribute to future generations. Since the creek stretches for miles, it is apparent that the separated populations of Mimulus have been limited on insect pollinators that are able to travel long distances to pollinate.

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Lab 4: Gel Electrophoresis/ PCR Cleanup

Date: September 17, 2019


Electrophoresis of PCR products

We first obtained our four PCR tubes and allowed them to thaw while we prepared the gel. We used a 10 µl micropipette to load sixteen 1 µl dye dots across a sheet of parafilm. Then, we pipetted 3-5 µl of PCR product into each dye dot. We used one additional dye dot as a negative control and two additional dye dots as a ladder. As we added our PCR product, we were recording the precise location of where each PCR sample went and who each sample belonged to. We set our micropipette to 5µl and used a filtered tip. We used the micropipette to suck up the dye dot and PCR mixture from the parafilm and transfer them into the wells of the gel. After all the wells were filled, the gel was run at 130 voltz for 30 minutes.


Clean-up of PCR products for sequencing – ExoSAP

I first obtained a strip of 8 new 0.2µl PCR tubes that I shared with my lab partner. We labeled each one with our individual sample codes without dividing the individual tubes. My sample codes were; EB01, EB02, EB03, EB04.  After we labeled the tubes, we proceeded to make the ExoSAP Master Mix for our table. We decided to run 18 PCR clean-ups to account for pipette errors. We first had to multiply each ingredient in our master mix by 18 to get the correct measurements to begin making the master mix. Our masters mix consisted of; 190.6 µl of H2O, 22.5µl of 10x buffer (SAP 10x), 7.92µl of SAP, and 3.96µl of Exo. All of these reagents were kept on ice throughout the process of making the master mix. The total volume of the master mix was 225µl. We then added 7.5µl of each of our PCR products into the new clean PCR tubes we had just labeled. Next, we used a 20µl micropipette to place 12.5µl of the mix into each individual PCR tube. The tubes were all sealed with their caps and placed in the thermocycler to start the EXOSAP program. Professor Paul removed the PCR tubes after 45 minutes of running the program, and placed them in a labeled tube rack and placed in the freezer.

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Lab 3: Plant Collecting

Date: September 10, 2019


This week for lab we took a trip to Marin Headlands where we were able to see the Mimulus guttatus plant up close and personal. We first walked along a trail that led us to creek that this plant occupied. We found that the Mimulus was found most closely to the water supply that the creek offered. I learned that these plants need moisture which is why we are most likely to find them in wet spots. You can see the beds of Mimulus in the photo below!

                             


We were able to take samples of this Mimulus gutattus for a future research project we will be doing in population genetics. We were given tubes that were filled with silica. The silica is used to dry the leaf and preserve its DNA for future use. The more dry the leaf becomes, the more DNA one is able to extract from the leaf which is why it was important for us to collect a small enough leaf sample that could be completely covered by the silica. You can see the tube and the leaf sample taken in the photo below!


Along the hike, I was able to learn various characteristics about this plant. I was pleasantly surprised to know that Mimulus guttatus is often used as a model organism because of its abundance of qualities. For example, Mimulus is able to grow in a diverse range of environments from a creek in the middle of the forest to the dry land surrounded by serpentine rock. In addition, Mimulus guttatus is able to grow rapidly and reaches maturity extremely quickly so it is able to reproduce at faster and helps researchers track generations of growth. Mimulus also has a genome that is less complex and smaller than many other plants which makes it easier and faster to sequence its genome. In the photos above, you were able to see a very vibrant and green version of Mimulus that was found along the creek that flowed through the forest. Below, I wanted to display a photo of the same plant from a neighboring environment that was much more dry and sat directly in the sun. As you can see in the photo, the Mimulus plant is able to adapt to the surrounding environment and produce seeds through pollinators. However, Mimulus growing in dry environments do not display the same vibrance of the same Mimulus that grow and produce seeds through water pollination.


The last site that we visited, just minutes away from the Marin Headlands trail, was the Serpentine rock which is shown below! I was able to learn and physically see that the state rock, Serpentine, was a green rock that was found everywhere in California where the tectonic plates collide underneath the land. The serpentine rock makes it extremely hard for plants to thrive to their full potential because the rock contains a multitude of metals. I was surprised to find out that despite how dry this land was, I was still able to see the Mimulus guttatus growing. It was not until then that, I was able to fully understand the importance of this plant and its use as a model organism for population genetics.


 

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Lab 2: Sushi DNA

Date: September 3, 2019


Collection:

I visited ‘We Be Sushi’ which is a small, family-owned sushi restaurant located at 538 Valencia Street, San Francisco, Ca. At the restaurant, I ordered four different types of sashimi so that the fish was raw and uncooked. I gathered one sample of king salmon, ahi tuna, sea urchin and albacore. I placed a tiny sample of each fish into its own separate 2.0 ml tubes. I immediately labeled each fish with a number that I assigned to each fish sample. I then went home and kept the samples on ice until lab on September 3rd.

DNA Extraction: 

Before starting the lab, I assigned each sample a unique ID code including the sample number and my initials. The king salmon was labeled ‘EB01’, ahi tuna was labeled ‘EB02’, sea urchin was labeled ‘EB03’ and the albacore sample was labeled ‘EB04’. Using gloves, I labeled one 1.5 ml locking lid microcentrifuge tube for each sample using the assigned ID code. I then took my samples off the ice and out of their tubes. I placed each sample into a plate which was divided into four sections for each fish. Using a razor blade, I cut the sample down even further to be able to get a sample that weighed between 2 and 10 mg. Using kim wipes and ethanol, I cleaned my razor blade and tweezers thoroughly as I switched over in cutting the different types of fish. I then took a piece of my king salmon, the ‘EB01’ sample, and properly weighed it using a zeroed scale and a sheet of wax paper. Once I got between the right weight range for my first sample, I was able to eyeball and cut the other samples to approximately fit within the weight range needed. Once each sample was weighed, they were each placed back on the plate until they were ready to be used. Taking the four labeled centrifuge tubes, I added 100 µl of Extraction Solution (ES) into each tube using a p200µl micropipette and unfiltered tips. Next, I added 25µl of Tissue Preparation Solution (TPS) to each tube using a 200 µl micropipette with a new unfiltered tip. I gently used the micropipette to mix the two solutions in the tube by sucking and releasing the mix up and down into the tube. Using tweezers, I placed each sample into its assigned tube with the mixture in it. I used an extremely small unfiltered disposable micropipette tip to mash the tissue sample at the bottom of the tube. The samples were each capped and put on a tube rack to sit and incubate for 10 minutes at room temperature. After the incubation period at room temperature, the samples were taken to incubate on a heat block at 95℃ for 3 minutes using a timer. After the 3 minutes, the tubes were taken out and each tube had 100µl of Neutralizing Solution (NS) added using a p200 micropipette. The solution was mixed by using a method called vortexing where the solution was placed on a vortex machine that shook the solution sample vigorously. I checked to make sure that the labeled remained prominent on each tube and then I was able to place each of these tubes on ice. 

Amplifying CO1 from Fish: 

Diluting gDNA: I first labeled four microcentrifuge tubes with ‘1:10’ and the unique ID code for each sample on the top and side of the tube. To start the 10x dilution for each of my gDNA samples, I first added 18µl of purified water to each tube that I had previously labeled using a p20 micropipette and the same filtered tip. Then, I added 2µl of the gDNA of each sample into the its matching tube using the p20 micropipette and a new filtered tip for every new sample. To mix the solution, I flicked the bottom of each tube to give it a gentle shake. 

Making the Master Mix: To make the master mix, I first had to use the volumes for the individual reactions of a PCR reaction and multiply them by the number of reactions and some extra. Since our group had 16 samples in total and we wanted to account for one negative control and one extra for good measure, I multiplied each volume by 18. I labeled a PCR tube with the name ‘master mix’ and began adding each reagent to the tube. Using a p200 micropipette and a new filtered tip for each reagent, I added 115.2µl of water, 14.4µl of forward primer, 14.4µl of reverse primer and 2µl of Tissue Extract (gDNA) to the PCR tube. I capped the tube until Professor Paul was able to come around and add the REDExtract-N-Amp PCR rx mix, that was not actually red, into our ‘master mix’ tube. While I waited, I labeled four PCR tubes with the same labels as my gDNA sample tubes and one tube as ‘negative control’ on the top and sides of the tube. I added 2µl of the 1:10 dilution of my gDNA to each PCR tube, except the ‘negative control’ tube. The professor was able to add 180µl of the PCR rx mix to the ‘master mix’ tube. Once the master mix was completed, I added 18µl of the mix to each of the five PCR tubes using a p20 micropipette with a filtered tube. The tubes were capped and placed on ice until each group was able to set up their PCR reactions. Once each group was done, I was able to put all five PCR tubes into the thermocycler and start the reaction that was estimated to take anywhere between 1.5-2 hours.