Samples were obtained Monday, August 26th, 2019 19:39 at Sakana Bune (5701 Geary Blvd, San Francisco, CA 94121) by ordering food and collecting samples in microcentrifuge tubes. Samples were stored in a freezer and thawed on September 3rd, 2019 for experimentation.
- p200 microcentrifuge
- 5 ml microcentrifuge tubes
- Razor blades/scissors/scalpels
- Heat block
Information about the samples was recorded in a table. Each sample was given a unique code (EVR01 – EVR04) and the species name it was listed as in Sakana Bune was recorded. (See Table 1) Gloves were obtained and put on. Labelled one 1.5 ml locking lid microcentrifuge tube for each of your samples with the unique ID code with Sharpie on the side and top of the tube. A paper plate was obtained to cut samples on. Samples were cut using a scalpel in order to cut away portions in contact with other ingredients. The scalpel was cleaned between cutting each sample. Samples were cut to a size of approximately 2 mg each and added to microcentrifuge tubes with their respective labels. 100 ml of Extraction Solution was added to each tube using a p200 µl micropipette with filtered tips. 25 ml of Tissue Preparation Solution was added to the microcentrifuge tubes with the 100 ml of Extraction Solution and was mixed through gentle micropipetting with a p200 µl micropipette and unfiltered tips. Disposable micropipette tips were used by hand to mash the samples in their respective tubes. Samples were incubated at room temperature for 10 minutes and 32 seconds. Samples were incubated on a heat block at 95o C for 3 minutes. Samples were removed from heat block and 100 ml of Neutralizing Solution was added to the samples using a p200 pipette and filtered tips and mixed by vortexing for 5 seconds each. Samples were then placed on ice.
The genomic DNA (gDNA) obtained through the above process was diluted. Labelled four microcentrifuge tubes with “1:10” and codes from Table 1 on the top and side of tubes. 18 ml of purified water was added to the tubes. Added 2 ml of gDNA to their respective tubes. Each tube was gently flicked to mix the solution.
A master mix was made to perform a PCR reaction (see Table 2). PCR tubes were labeled with the codes from Table 1 on the top and sides. 2 ml of the 1:10 dilutions of gDNA was added to each respective PCR tube, except for the negative control tube. 18 ml of the master mix was pipetted into each PCR tube and the negative control. Samples were put in the thermocycler and placed in the freezer when the cycling was complete. Settings for the thermocycler were as follows:
94o C – 4 min (initial denaturation)
30 cycles of:
94o C for 30 sec (denaturing)
52o C for 40 sec (annealing)
72o C for 1 min (extension)
72o C for 10 min (final extension)
10o C hold
The results of this lab can be found in Figure 1 under the heading “Triple Threat”.
Reagent Volume (1x) Master (volume x 13)
Water (PCR Quality) 6.4 ml 83.2 ml
REDExtract-N-Amp PCR rx mix 10 ml 130 ml
Forward Primer 0.8 ml 10.4 ml
Reverse Primer 0.8 ml 10.4 ml
Tissue Extract (gDNA- 1:10 dilution) 2 ml ———
Total Volume 20 ml 234 ml