I collected my raw fish samples from Kufuya Japanese Restaurant at 7001 Geary Blvd, San Francisco, CA at 7:30 PM on Monday 2 September 2019. I ordered a plate of nigiri (raw fish on top of rice) with three different species of fish: yellow tail, white tuna, and red snapper. I cut a small piece of each species of fish and stored each piece in a 2.0 ml tube provided in class. I labeled each tube with a unique code and recorded which code corresponds to each species of fish. I stored the tubes with fish samples in my home refrigerator over night, and then transferred them to an ice bucket in lab the following day.
Figure 1. Photograph of assorted nigiri plate as served. Fish species ordered (from left to right): yellow tail, white tuna, red snapper
Reagents: I used a commercial DNA extraction kit (Sigma REDExtract-N-Amp Tissue PCR Kit)
-Extraction Solution (labeled ES)
-Tissue Preparation Solution (labeled TPS)
-Neutralizing Solution (labeled NS)
-1.5 ml microcentrifuge tubes
1. I recorded the ID code and species common name for each of my samples on the “Animal Tissue DNA Extraction” data sheet. I gave each sample a unique ID code, starting with my initials (NI) and then a number (01-03). I recorded the species name that my samples were labeled as on the menu. The codes correspond as follows:
Unique ID Code Restaurant Species Name
NI 01 Yellow Tail
NI 02 White Tuna
NI 03 Red Snapper
2. I wore gloves and other appropriate PPE for the remainder of the DNA extraction procedures.
3. I used a sharpie to label one 1.5 ml Locking Lid microcentrifuge tube for each of my samples with the unique ID code. I wrote the unique ID code BOTH on the side and on the top of the tube.
4. I took each sample out of its tube, and placed on a labeled paper plate to cut out a small piece. Using a scalpel, I carefully cut out a small square of fish tissue. I made sure to cut away any portion that was in contact with the rice and used tissue from inside the sample. In between samples, I cleaned the scalpel with Ethanol, and cut specimens on different parts of plate, which were labeled.
5. I put a weigh boat on a scientific scale, and zeroed the scale. Then I added a sample of yellowtail to weigh the sample. I cut the sample smaller, and reweighed until I had a piece that was approximately 10 mg. I then cut similar size pieces of the other samples, using the yellow tail sample to estimate the weight of the subsequent samples.
6. I added each 10 mg sample to its corresponding extraction microcentrifuge tube using forceps, cleaning the forceps in-between each sample.
7. I added 100 µl of Extraction Solution (ES) to each of my labeled sample tubes using a p200 μl
micropipette and unfiltered tips.
8. I added 25 µl of Tissue Preparation Solution (TPS) to each of the microcentrifuge tubes with the 100 µl of Extraction Solution (ES) and micropipetted up and down to mix using a p200 μl micropipette
and unfiltered tips.
9. I used disposable non-filtered pipette tips to gently mash my tissue samples by hand.
10. I incubated the samples at room temperature for 10 minutes.
11. I then moved my samples to the heat block to incubate at 95oC for exactly 3 minutes.
12. After 3 minutes, I removed the samples from the heat block. I then added 100 μl of Neutralizing Solution (NS) to each tube using a p200 pipette and filtered tips. I mixed each sample by vortexing on a vortex machine for ten seconds.
13. I then double-checked that the labels had not been rubbed off the tubes, and put the samples in an ice bucket.
Amplifying CO1 for PCR:
Diluting the gDNA
To make a 10x dilution of the gDNA:
1. I labelled a microcentrifuge tube with “1:10” and the unique ID code for each sample from the previous steps. I wrote this on the top and on the side of each of the 3 tubes.
2. I add 18 µl of purified, filtered, sterile water to each of the labeled dilution tubes.
3. I added 2 µl of the gDNA (from the previous steps) of each species to the dilution tubes.
4. I gently flicked the tubes with my finger to mix the solutions.
The PCR reaction
Each of my PCR reactions included the following reagents and volumes:
Water (PCR Quality – autoclaved, filtered) 6.4 µl
REDExtract-N-Amp PCR rxn mix 10.0 µl
Forward Primer FbcF 0.8 µl
Reverse Primer FbcR 0.8 µl
Tissue Extract (gDNA) (1:10 dilution) 2.0 µl
Total Volume 20 µl
To set up multiple PCR reactions under the same conditions, including my 3 samples as well as the samples for my lab-mates, we made a master mix that included the combined volumes of all of the reagents for multiple reactions, except for the diluted tissue extract. This strategy improves success by minimizing errors due to pipetting small volumes multiple times.
To make enough master mix for the whole table, we used the volumes for the individual reactions (listed
above) multiplied by the number of reactions, plus some extra just in case. Our table had 13 samples, one negative control, and we wanted to make sure we had a little extra. So we would multiplied the volumes times 13 + 1+ 2 = 16.
A negative control is all the reagents required for a PCR reaction, but NO gDNA template. Thus, if anything is amplified in the negative control, we know there is a source of contaminating gDNA, other than the samples we are trying to PRC amplify.
The additional 2x reagents are needed to account for minor errors in pipetting that could result in
not having enough total volume in the master mix for all individual reactions.
The master mix recipe is:
Reagent Volume (1x) Master (16x)
Water (PCR Quality – autoclaved, filtered) 6.4 µl 102.4 µl
REDExtract-N-Amp PCR rxn mix 10.0 µl 160 µl
Forward Primer FbcF 0.8 µl 12.8 µl
Reverse Primer FbcR 0.8 µl 12.8 µl
Tissue Extract (gDNA) (1:10 dilution) 2.0 µl —-
Total Volume 20 µl 290 µl
1. I wrote the labels of my gDNA sample on PCR tubes in sharpie (on the top and on the side just below the lid).
2. The master volume of each reagent above was added to a tube labelled “MM” for master mix, except for the REDExtract-N-Amp PCR rxn mix.
3. I added 2 µl of the 1:10 dilution of each gDNA dilution to each of the labeled PCR tubes, except the negative control, changing pipette tips in-between each sample.
4. I added the REDExtract-N-Amp PCR rxn mix to the master mix.
5. I then Pipetted 18 µl of the master mix into each of my PCR tubes, including the negative control, changing pipette tips in-between each sample.
6. We set these PCR reaction tubes on ice next to the thermocycler until all the PCR reactions were set up. We then put the PCR tubes (all samples and the negative control) in the thermocycler and started the reaction, which takes between 1.5-2 hours.
7. When the PCR reactions were finished with the therm-cycling (see settings below), they were placed in the freezer.
Settings for the thermocycler:
94oC – 4 min (initial denaturation)
30 cycles of:
94oC for 30 sec (denaturing)
52oC for 40 sec (annealing)
72oC for 1 min (extension)
72oC for 10 min (final extension)
Figure 2. Photograph of my lab table’s DNA extraction gel electrophoresis results. My three samples are labeled NI01, NI02, and NI03.