Professor John Paul
Lab 2 Entry
The Sushi Test
On September 4th of the year 2018, I walked to Lucky Fulton Market at 1750 Fulton Street, located in the city of San Francisco.
At around 2:18 PM, I purchased three types of uncooked fish that were displayed on ice at the store for a total of $9.44.
I bought one fillet of fresh Wild Pacific Rock Cod (USA) for $6.99/lb. A small sliver of the fish (about the size of a fingernail) was then cut off and inserted into a 2.0 ml tube and labeled/recorded as “1” on the tube.
I bought one fillet of fresh Wild Dover Sole (USA) for $7.59/lb. A small sliver of the fish (about the size of a fingernail) was then cut off and inserted into a 2.0 ml tube and labeled/recorded as “2” on the tube.
Lastly, I bought a cut of Wild Ahi Tuna (Indonesia) for $6.99/lb. A small sliver of the fish (about the size of a fingernail) was cut off and inserted into a 2.0 ml tube and labeled/recorded as “3” on the tube.
Thought: While at the grocery store, there were several other options that were available for purchase; however, these were among the few that had a descriptive detail of the type of fish being sold.
As soon as the samples were collected from each of the three fish types and correctly labeled on the tube that they were put in, the tubes were placed into a freezer until the next day when lab for molecular ecology would take place.
The next day on September 5th at around 12:45, I brought the three tubes containing the fish to the lab. I began the lab by extracting the DNA from the fish. First, I labeled my three species of fish that was provided by the grocery store with their corresponding identification number and my initials (Ex. Rock Cod=CP01) on a piece of paper that would be used for later purposes regarding the fish’s DNA Barcode Species name. I retrieved three 1.5 ml Locking Lds and labeled one of them “CP01”, another “CP02”, and the last one “CP03” with a sharpie. I then put gloves on and used a scalpel to carefully cut each of the fish types into tiny pieces that measured a weight somewhere in between 2 to 10 mg on a scale. I was sure to use ethanol to clean my scalpel before cutting a new type of fish in order to prevent any chance of contamination. After cutting the fish into the correct approximate weights, I then put the fish aside on the table on clean square tissues. Next, I used a p200 micropipette with unfiltered tips to put 100 microliters of Extraction Solution (ES) into each of my 1.5 ml Locking Lds tubes. (During my first attempt I misunderstood the instructions and accidentally put my fish samples into the 1.5 ml tubes and then added the Extraction Solution with it. I then had to cut new samples of fish and start over to ensure I would succeed in extracting my fish DNA). Using the same pipette but a different tip, I then added 25 microliters of Tissue Preparation Solution (TPS) into these same tubes and slowly drew up and released the contents inside each of the 1.5 ml tubes to allow mixing to occur. I then used forceps to insert my fish samples into the mixture of each 1.5 ml tubes, making sure I used ethanol to clean the forceps after each fish to prevent contamination. By mashing the fish inside the mixture with a sterile unfiltered pipette tip, I was able to decrease the size of the fish sample. Each of the 1.5 ml tubes was then left on the lab tables to incubate at room temperature for ten minutes and then transferred to a heat block for further incubation at 95 degrees Celsius for precisely three minutes. I then used a p200 micropipette and filtered tips to put 100 microliters of Neutralizing Solution (NS) into each of the 1.5 ml tubes. I vortexed these three 1.5 ml tubes for about 5 seconds each and then put them on ice.
For the next part of the lab, I conducted an agarose gel electrophoresis for my three types of fish DNA. I began by acquiring pre-made 1% agarose gel from my professor. After making sure the top of the gel was near the red electrical connector and that the level of 1x TAE buffer barely covered the height of the wells in the gel, I got a piece of parafilm for my lab partners at my table. One of my partners (Kayla) used a p20 micropipette to drop 2.0 microliter droplets of Loading Dye in four rows of three onto the parafilm (our lab table had four people, each with three different fish samples) and labeled each partner’s row and identification number near the droplets. Kayla continued to use the same pipette, but now with a new tip and added 3.0 microliters of each of our genomic DNA to each of our three existing droplets of Loading Dye. I then took over and used the same pipette to transfer each of the now 5.0 microliter droplets from the parafilm to the specific gel wells that our group categorized and labeled. We then put a lid over the gel box and hooked up the red wires with the red connector and the black wires with the black connector. We set up the volts to 145, pressed the button to “run” the gel, and made sure that the gel electrophoresis was indeed undergoing. After about 15 minutes, our group retrieved the gels and brought them to the machine where the existing Gel Red in the gel would allow special imaging of the DNA lengths to be developed and taken. Once our professor saved our group’s gel images, we recycled our gels so that they could be reused.
On the top row going from left to right (Kayla, Carter) and on the bottom row going left to right (Peter, Jinwoo). Both are the same images of my group’s gel; however, the image on the right has the colors inverted to give a different contrast and visual of the lengths of our fish samples’ DNA. All of our samples were successful in the DNA extraction and gel electrophoresis.
After running a gel electrophoresis for our fish samples’ DNA, amplification of CO1 from fish was the next goal. I started by labeling three centrifuge tubes with their specific identification number and “1:10” (Ex. CP01 1:10) on the top and side. I then added 18 microliters of purified water in each of the tubes with a micropipette. Next, the micropipette tips were changed and 2 microliters of my gDNA were added to the tube. By gently tapping the tubes on the table, I was able to dilute my gDNA. I then acquired three different PCR tubes and labeled them with their specific identification number (Ex. CP01) on the top and side. I added 2 microliters of the 1:10 dilutions of gDNA from the centrifuge tubes to the corresponding PCR tubes with the correct identification number (Ex. CP01 1:10 centrifuge tube contents transferred to CP01 PCR tube). I then acquired the master mix from my lab partner (Jinwoo, who made it for our table) and changed pipette tips to then transfer in 18 microliters of the master mix into each PCR tube (Our group decided on making a master mix that had a volume x 18, to ensure more than enough was available for each person at the table just in case somebody transferred too much master matrix into their PCR tubes. I then put my PCR tubes into the ice container until all students in the lab were finished. Once everyone was ready, I placed my PCR tubes into the thermocycler along with everybody else’s PCR tubes. Our professor then turned on the thermocycler and began the process of amplifying the C01 from my fish DNA.
Thoughts: I felt a little rushed at times during the lab project. I tend to take a long time double and even triple checking measurements and tools to ensure that I am following the procedure correctly. Often, this results in me being one of the last to complete parts of the lab compared to the majority of my fellow classmates.
On September 13th, I will continue with this DNA Barcoding project on my three fish samples and observe the results of my PCR tubes that I put in the thermocycler the last lab on September 5th.