Authorgmsalgado

Using Geneious Software to compare animal tissue DNA extractions

In this lab, we were able to download a program, Geneious, which allowed us to access the sequences produced by our DNA extractions of animal tissue from sushi. From the 4 samples of DNA, only 2, GS01 and GS02, were viable to produce sequences from both the forward and backward primers. Starting with GS01, the forward primer sequence had an HQ (High Quality) of 74.2% and the reverse primer sequence had an HQ of 25.7%.Using the DeNovo feature, we were able to combine the forward and reverse primer sequences and edit the sequence to produce a consensus sequence with an HQ of  81.9%. This number was produced only after editing sequences, which consisted of cutting off the ends of the sequence and erasing or fixing single bases. GS02 was subjected to the same DeNovo feature and editing process to produce a consensus sequence with an HQ of 78.8%.

We then were able to BLAST (Basic Local Assignment Search Tool) each of our consensus sequences, which allowed us to search a database for highly similar sequences. We were able to determine how well the sequences matched based on the Grade of the sequence. We selected 5 species to compare the consensus sequences to. Using this tool, I was able to conclude that the sample labeled GS01 was most likely Thunnus Albacares, which is a yellowfin tuna meaning the sushi market was in fact selling tuna. Sample GS02 was also analyzed using the BLAST tool and after comparing the consensus sequence it was determined to most likely be Seriola quinqueradiata, which is a yellowtail fish as advertised in the sushi market.

While the sequences were very similar, there were polymorphisms. For GS01, there were 4 polymorphisms which resulted from editing since some bases were erased due to convoluted fluorescence. There were also may bases that were not definitively determined, thus the were discrepancies in those as well. Base 511 had GS02 consensus sequence of a T nucleotide, however, there the rest of the species had a consensus in which there was no base there. There was also a similar situation at base 498. At base 121, the fluorescence was confusing and caused a polymorphism which is depicted in figure 2.

 

Figure 1: This is a GS01 polymorphisms in which a T nucleotide was incorrect.

Figure 2: GS01 had a consensus sequence that produced a T nucleotide, however the species consensus was a C.

When GS02 was compared to 5 other sequences, there were 22 polymorphisms. Although there were many polymorphisms, this was mostly due to including a 2 species that were not as similar to the sequence that were included in the comparison. However, when GS02 was compared to just the Seriola quinqueradiata, there were no polymorphisms. An example can be seen in Figure 3.

Figure 3: A GS02 polymorphism when comparing 5 other species.

Field Trip 9/23/19

A Mimulus guttatus by a water fountain and exposed to large amounts of sunlight

On our field trip, we traveled to Muir Beach, near Mountain Tamalpais. On this trip, we were able to see multiple populations of Mimulus guttatus in different environments. Due to the different environmental pressures, these populations demonstrated different traits that correlated to adaptive pressures.

These mimulus are not yet matured, but will most likely flower due to their environment

For example, the first mimulus plants we saw were exposed to high amounts of sunlight and had constant water supply from a fountain. This caused there to be flourishing populations that were also flowering, thus signaling a readiness of maturity and to pass on genes to next generations. The plants in this area were healthy enough and in a good position, so they flowered, and bees most likely acted as pollinators for these plants since the provide an ideal landing platform for bees and red spots on the petals that could be attracting the bees. We spoke of the likeliness that a pollinator would travel far to spread the mimulus’ pollen, and determined that the mimulus guttatus that were closer would be receiving those genes rather than further ones. This would mean that there is a high probability of inbreeding, which can be dangerous to the gene pool. We were also able to see what mimulus looks like in its primary stages of life in this area as another population began to flourish. It is also worth mentioning that the mimulus populations were usually found around horsetail and ferns.

The mimulus here comes from a flowering plants and demonstrates the ideal bee landing petals and red spots

We then traveled to a more shaded part of the terrain where a creek was found. After traveling up the creek, a population of mimulus guttatus was found, but the populations was not flowering. The reason for the lack of flowering can be explained through the location of the plants. They were located in a shaded creek area that would get flooded in a few months time and had very little pollinator traffic, thus, along with shortening of the days, the mimulus reacted to these cues by not reproducing. These populations are called sink populations and they will not be participating in breeding with any other populations.

These mimulus are not flowering because of the lack of reproductive cues

These different populations are most likely not to be very connected, but it all depends on the distance that pollinators travel and the seed dispersal that occurs. Mimulus guttatus is a very diverse plant and continues to demonstrate the adaptive capabilities it possesses.

Gel Electrophoresis and PCR Cleanup

In the second part of of the DNA extraction lab from animal tissue derived from sushi samples, our lab group ran a gel electrophoresis and PCR clean up. The first step in this process was obtaining our saved PCR tubes and letting them thaw at room temperature, and then the samples were placed on ice. In order to use our samples in gel electrophoresis, we pipetted 16 dots of dye on a sheet of parafilm. Each dot was approximately 1.5 microliters. We then pipetted 3 microliters of each PCR product into its own dot while changing pipette tips in between each dot. Each member was responsible for their own PCR products. After each dot of dye had PCR product in it, we set a pipette to  5 microliters and loaded each dot into our gel. We ran the gel at 130 volts for 30 minutes.

While our samples were running gel electrophoresis, we started our PCR cleanup. We began by carefully labeling new 0.2 microliter PCR tubes with our sample codes. I labeled my tubes with GS01, GS02, Gs03, and  GS04. We proceeded to make an ExoSap Master mix by mixing 211.8 microliters of pure water, 25 microliters of 10x buffer (Sap 10x), 88 microliters of SAP, and 4.4 microliters of Exo. We were able to do this successfully eventually, but initially, we did not use the correct amount of buffer or SAP, thus we had to redo our master mix. We mixed our successful master mix by holding the tube of master mix and waving our arm left and right on a horizontal plane, multiple times.

We pipetted 7.5 microliters of each PCR product into a clean, labeled, 0.2 microliter PCR tube. We also added 12.5 microliters of the master mix into the labeled PCR tube and then placed all PCR tubes into a thermocycler and started the EXOSAP program for approximately 45 minutes. When the program was completed, our professor placed the PCR tubes in a labeled rack and then they were placed in the freezer.

Field Trip on 9/10/19

Mimulus guttatus

On this field trip, our class traveled to Mountain Tamalpais where we were able to view the drastically different environments in which the species, Mimulus guttatus grows. We began the field trip by observing the shaded creek in which there was a large population of lively Mimulus guttatus. The environment consisted of a water bed from the creek and essentially no sunshine because of the shade created by all of the trees. This demonstrates how mimulus can grow without much sunlight. We were then able to collect samples of the species by pulling off the newest leaves from mimulus species that were at least 2 meters away from each other. The samples were places in a plastic tube with silica that would prevent moisture from accumulating and damaging the DNA sample.

This is the mimulus that I picked from, they were located in a shady creek

After collecting the samples, we traveled a short distance to a very dry area in the middle of a field where all plant life was very much exposed to the sun. In this area, there was a dried up creek that had some dried Mimulus guttatus that was previously seen alive. It is evident that the mimulus would have gotten the needed water from the creek that was there and was also equipped to handle large amounts of sunlight. We were also able to view the inside of a seed pod and view the seeds that species carries. They were incredibly small and thus it could be inferred that animal dispersal from feces or other matters were not a likely form of seed dispersal.

This is the area where the mimulus was dried because of sun exposure and a dried up creek.

 

These are the seeds from the mimulus guttatus, they are very small.

Next, we visited an area that plentiful with serpentine rock where plants have difficulty growing because of the heavy metals that come with the serpentine. The mimulus that were around this area were annual plants, meaning that the plants only grew once a year.

We then walked a further distance and reached an area in which there were many dry plants and sunlight exposure, but there was also a small creek that flowed in a valley. While the plants around the area were mostly dry, there was live mimulus guttatus where the stream was located. These species were flowering and the yellow color as well as the landing space of the petals suggested that bees were the pollinators that aided in seed dispersal of mimulus guttatus.

At the conclusion of the field trip, students were able to determine that Mimulus guttatus has been able to adapt to various environments. This can be due to gene flow that is transferred by bees and other pollinators that allows the plant to have certain traits that help the plant adapt.

DNA Extraction from Animal Tissue

 

This picture shows the sushi at the Japanese market place

On September 2nd, 2019, I purchased packaged sushi from the Japanese grocery store, Nijya Market in San Francisco. On package was labeled to contain sushi that contained raw salmon and raw mackerel. Another package had raw tuna rolls, and the other had raw yellow tail. Pieces of the animal tissues from each fish were then placed into screw-cap microcentrifuge separately from each other and put on ice.

The image above shows myself removing the rice and other obvious ingredients from the raw tuna.

In order to extract DNA from fish tissue derived from sushi, it was first necessary to record the animal tissues that were collected on a data sheet. This included tuna, yellow tail, salmon, and mackerel. Each sample was given a unique code: tuna was labeled GS01, yellow tail was labeled GS02, salmon was labeled GS03, and mackerel was labeled GS04. Next, we put on gloves and labeled four 1.5 ml locking lid microcentrifuge tubes, one for each sample, with sharpies and writing on both the side and the top of the tube.

We cut a small piece of each sample using a scalpel and a paper plate as a surface. In order to determine the size of the sample piece that was cut, one of the samples was cut and weighed to 10 mg. The size of the 10 mg sample was then compared to other samples in order to create uniformity among the size of the samples.

Each sample was placed into its own 1.5 ml microcentrifuge tube. Before the addition of the sample piece, 100 microliters of Extraction Solution (ES) was added using a 200 micropipette and an unfiltered tip. 25 microliters of Tissue Preparation Solution (TPS) were also added using a 200 micropipette and unfiltered tips. The solution of ES and TPS was mixed by micropipetting up and down gently. The animal tissue sample was then placed in the corresponding labeled tube using forceps. The samples were mashed in each tube using a disposable non-filtered pipette tip. The samples in the tubes were then left alone at room temperature for 10 minutes.

The samples were placed on a heat block and incubated at 95 degrees Celsius for 3 minutes, using a phone as a timer. The sample was then removed from the heat block and and 100 microliters of Neutralizing Solution was added using a 200 micropipette with a filtered tip and mixed by vortexing. After this was completed, the samples were put on ice for approximately 4 minutes.

In order to assure good function of PCR, the gDNA was diluted by 10-fold. To do this, we labeled a microcentrifuge tube with “1:10” and the corresponding unique code on the side and top of the tube. We then added 18 microliters of purified water in the labeled tube along with 2 microliters of the gDNA that we extracted from the collected animal tissue. We also used the vortex machine to mix the solution.

We proceeded to make a master mix for the PCR that included 128 microliters of PCR quality water, 200 microliters of RED Extract, 16 microliters of forward primer, and 16 microliters of reverse primer. The volume was determined by multiplying the original volumes by 20 in order to accommodate for all members at our lab table and have extra for a negative control. We then used the master mix and placed 18 microliters of the master mix along with 2 microliters of our gDNA into a PCR tube. This process happened with each different gDNA while switching pipette tips in between each sample.

We wrote the labels of what gDNA it was on each PCR tube on the side and the top of the tube. The tubes then were placed in the thermocycler along with the negative control. Once the PCR reactions were all set up, the reaction ran for 1.5-2 hours. The PCR reactions were then placed in the freezer when the cycling was complete.

We are group Turtles, and the samples that are specifically mine preceded with “GS.” All samples were successful in producing a product.

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