Date: September 3, 2019
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.
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.