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.