Lab Entry 6 – Geneious Gene Sequencing and Analysis

With the completion of DNA replication and running multiple PCRs, results of gene sequences were received in which analysis could take place. A program, Geneious, allows for this analysis by comparing the forward and reverse sequences of data found for each of the samples. All three of my samples produced sequences with high HQ% values. With all six of the forward and reverse sequences placed into a file on Geneious, a comparison could be made for each of the three samples.

First, the two matching sequences were selected and then ran through De Novo Assembly to piece the sequences together to match each other as accurately as possible. The program runs the forward and reverse sequences in the same direction for a comparison to occur. Any base pairs that were unable to be sequenced properly were deleted while some base changes were altered in order to match the other sequence based on the level of accuracy produced by the program. Once the sequences were trimmed and corrected to match each other, the sequence was compared to other sequences withing the Geneious  in order to determine the origin of the sample. The program lists a multitude of potential matching sequences, providing a percentage of how closely they relate to each other. The sequence with the highest sample can be assumed to have the same DNA as the sample tested resulting in the genetic identification of the species of sample.


1). All of the samples were correct when comparing sequence identification to what the samples were marketed as in the restaurant. Chinook salmon, yellowfin tuna, and yellowtail were all correctly identified from the sequencing performed.

2). The salmon sample had no polymorphic sites when comparing my sequences to the sequences of the best matching species. The tuna sample had quite a few polymorphic sites where there were different base pairs at the same location. The yellowtail sample had two polymorphic sites near the end of the sequences but matched perfectly other than those two differences.

3). For the third part, 27 sequences were collected to go along with the three sequences from my samples for a total of 30 sequences to compare with. The sequences consisted of ray-finned fish species (Actinopterygii) and a few shark and ray species (Chondrichthyes).


Field Trip II

Lab Entry 5 – Field Trip II


For the second filed trip the class took, we ventured to the Limantour Trailhead area in Point Reyes National Seashore. This location was approximately 1.5 hours north of San Francisco via driving. Lupinus arboreus was found on the trail to the beach with a distinct difference that the plants found in the Half Moon Bay area from the last field trip taken. Lupinus arboreus at this northern location consisted of flowers with a purple color rather than yellow as seen in Half Moon Bay. This was the only visible difference between the plants in the north from the south. In addition to viewing our Lupinus arboreus, wildlife was also seen including seals and humpback whales. Samples of Lupinus arboreus were collected prior to the field trip by the Professor Paul .


Gel Electrophoresis

Lab Entry 4 – Gel Electrophoresis

A template organizing where the samples were placed was provided in which each of the samples for the table was designated a specific lane to be place in. The gels being used have two rows with 8 lanes in each. The top row of gel was occupied by other table-mates and a ladder while the bottom row consisted of my three samples and another ladder. The gel was loaded in accordance to this template.

gDNA Electrophoresis

With a 1% agarose gel with GelRed already cast, the gel tray was placed into the gel box with the top of the gel at the negative (black) end of the electrical connector. 1x TAE buffer was poured into the gel box so that there was enough to cover the gel by a couple millimeters.

On a piece of parafilm, 2.0 μl Loading Dye was dropped for the number of samples being used. In our case, we placed 12 drops on the parafilm to be used for each of the samples for the table. Approximately 3.0 μl of gDNA was added to the Loading Dye for each of the samples. This was done until all dots were completed with Loading Dye and gDNA.

The pipette was readjusted to 5.0 μl and then each drop was pipetted into the designated well on the gel for all of the samples of gDNA. Once all of the wells were filled with their designated samples, the lid was placed onto the gel box with positive and negative charges aligned and then turned on. The volts were set to 145 and the gel ran for approximately 16 minutes before the power was turned off.

The gel was then imaged using the Gel Doc EZ Imager.

Making 1% Agarose Gel

For the gel used for the PCR electrophoresis, the gel previously used for the gDNA run was melted down in a microwave to be reused for the PCR run. The gel was placed into a beaker and placed in a microwave for approximately 25 seconds, allowing the solution to bubble for about 10 seconds. To the liquid, another 1 μl of GelRed was added to the beaker in case of any loss. The gel was then poured into the casting rig set up to make the gel with the two rows of wells using combs. The gel was left to harden for about 10 minutes to be used for the PCR electrophoresis run.

PCR Electrophoresis

For the PCR electrophoresis run, the same procedure was performed as for the gDNA electrophoresis run. All of the measurements were the same as well as the entirety of the process of dying and placement of samples on the gel. The only difference was the addition of a negative control that was made in the previous post which was then placed into one of the wells on the gel. Voltage and time for the electrophoresis run were the same as for the gDNA run.

Exo-Sap Clean-up

For the clean up process, we used RA02, RA03, JPCC, JPCD, JW01, JW02, & JW03. These lanes had the best results for banding based on the computer imaging of the PCR gel plate. A master mix of ExoSap solution was created for all of the table-mates. Enough solution was created so that 9 clean-ups could be performed from the best samples and extra solution in case it was needed. The master mix consisted of 95.31 μl water, 11 μl 10x buffer (Sap 10x), 3.96 μl SAP, and 1.98 μl Exo. 7.5 μl of PCR product was placed into 0.2 μl PCR tubes for each of the samples in addition to 12 μl of the ExoSap master mix. Once all tubes contained the necessary amounts of products, they were placed into a thermocycler to undergo the EXOSAP program which takes approximately 45 minutes.


DNA Extraction from Animal Tissue

Lab Entry 3: DNA Barcoding

Extraction of DNA:

Information was first taken to ensure that labels were correct between tubes and fish samples. Three varieties of fishes were collected and assigned the labels JW01, JW02, and JW03 for salmon, yellowtail, and tuna, respectfully. Gloves were used throughout the entirety of the procedure. Fish samples were derived as stated from a previous lab entry. The Sigma REDExtract-N-Amp Kit was also used for this lab which provided various components and solutions needed for extraction and amplification (next part).

Three 1.5-ml screw-cap centrifuges were labeled for each of the samples collected. Each of the samples were cut into small pieces (approx. 2-10 mg) on a plate using scalpels and forceps while cleaning the instruments between each sample with Ethanol in order to prevent cross contamination.

Using a p200 µl micro-pipette and unfiltered tips, 100 µl of Extraction Solution (ES) was added to each of the labeled tubes. 25  µl of Tissue Preparation Solution (TPS) was then added to each of the labeled tubes as well. To mix each of the solutions, the micro-pipette was pressed up and down. Solutions were derived from the kit mentioned earlier.

Each of the fish samples was then finally added to the corresponding microcentrifuge using forceps. Using hands, rather than the micro-pipette, a non-filtered pipette tip was used to gently mash the tissue sample. The sample was then left to incubate for 10 minutes at room temperature.

The samples were then moved to a heat block where they incubated at 95°C for 3 minutes which was measured using a timer. Samples were removed as close to the 3 minute mark as possible. Once removed from the heat block, 100 µl of Neutralizing Solutions (NS) was added using a p200 pipette and filtered tips and was then mixed by vortexing. The samples were then checked to make sure the labeling remained intact and then put on ice.

Amplifying COI from Fish:

For the amplification portion of the lab, the Sigma REDExtract-N-Amp Kit was used again. To amplify the genomic DNA (gDNA) that has been isolated from the previous part, PCR was conducted using Taq, dNTPs, and buffers provided from the kit.

DNA was first diluted 10-fold for optimal success when running PCR. A microcentrifuge tube was labeled “1:10” plus the name of the sample which also had my initials in the name. 18 µl of purified water was added to the tube followed by adding 2 µl of gDNA into the tube. The tube was gently flicked with finger to mix the solution.

A master mix was then made that contained all of the reagents being used for the solution when running the PCR that was shared among the lab table. In order to have the necessary amount of reagents for all of the table-mates, the master volume ratio was multiplied by 14 so that everyone would have enough solution. The master mix used had a total volume of 280 µl consisting of: 89.6 µl water, 140 µl REDExtract-N-Amp PCR rx mix, 11.2 µl forward primer, and 11.2 µl reverse primer. Extra reagents were added following the ratio for the purpose of ensuring that everyone would have enough solution for each of their gDNA samples.

For the three gDNA samples, three PCR tubes were labeled with corresponding labels on the top and sides of the tubes. 2 µl of 1:10 diltuion of gDNA was added to a PCR tube individually for each of the three samples. One table-mate created the negative control to be used during PCR that contained no gDNA in which case, the PCR tube remained empty for now. At this stage is when it was determined that a faulty pipette had been used when measurements were made. The pipette was incorrectly collecting solution amounts that may affect the results of PCR. The pipette was used earlier in the lab as well which may alter the outcome since ratios and amounts of reagents could have incorrectly been obtained. However, the master mix was not affected by the use of the faulty pipette and therefore, was used in the next step.

Once all of the tubes had their gDNA, 18 µl of the master mix was added into each of the PCR tubes, including the negative control tube. Pipette tips were changed between each of the samples. The samples were mixed via vortex and then placed on ice until all samples of lab-mates were ready for the next step.

All the PCR tubes including the negative control were then placed into the thermocycler and the reaction was started. The thermocycler takes approximately 1.5-2 hours and once completed, samples were placed into a freezer.

Settings for thermocycler:

  • 94°C – 4 min (initial denaturation)
  • 30 cycles of:
    • 94°C for 30 sec (denaturing)
    • 52°C for 40 sec (annealing)
    • 72°C for 1 min (extension)
  • 72°C for 10 min (final extension)
  • 10°C hold

The remainder of the lab will be conducted in the following lab session with a different lab post.


Field Trip I

Lab Entry 2 – Field Trip I

For the first field trip of the semester, the class headed south of San Francisco to the Half Moon Bay area in order to collect plant samples, specifically the Lupinus arboreus within the legume family. In order to retrieve these plant samples, a field trip was taken approximately 42 miles to the south to Pescadero State Beach where we encountered our first collection of plant samples. We then headed north to Cowell Ranch Beach which is about 30 miles south of USF. At each of the locations, five samples were collected from five different lupine bushes to provide more data to be used when analysis occurs. The plant samples were placed in tubes that contained silica to dry out the tissues for the purpose of preserving the DNA for a longer period of time. Another collection field trip will occur in which more of the lupine plants from an area north of USF rather than south will be collected which will then be used to compare to each other under experimentation. The lupine collected in the Half Moon Bay Area all consisted of yellow flowers which can be seen in the images provided from the field trip.



Sushi Collecting!

Lab 1 Entry – Sushi Collecting

For the first assignment, raw fish samples were to be collected in the form of sushi and nigiri in my case. The fish samples were collected from Tsunami Sushi in SOMA in which Hamachi-Yellowtail and Sake-Salmon (pictured with two pieces) nigiri dishes were ordered in addition to a spicy tuna roll (pictured on left of the two sushi rolls while the other roll was a spider soft-shell crab roll). The menu stated that many of the fishes served in the restaurant were acquired from the famous Tsukiji Market in Japan or locally sourced. The samples were placed into three separate tubes labeled with the type of fish and tehn placed into a freezer in order to preserve the samples. This leads me to believe that the fishes being served at Tsunami are accurately labeled and marketed to the customer but after DNA barcoding, the truth will be revealed.


Tsunami Sushi – 301 King St, San Francisco, CA 94158