For the DNA extraction procedure, I used 2 copies of one sample of fish labelled “Red Drum” that I purchased at the 88 Manor Market located near my apartment in San Leandro, CA. Tissue from the region near the gills was collected in 2 vials and stored on ice until the procedure. The samples were stored on ice in my freezer until the day of the labs, when they were needed. From the defrosted samples, I removed roughly 20 mg of tissue for each on the 2 copies and added them into a 1.5 mL reaction tube labelled with their designations (JK01 and JK02, respectively). To each tube, 100 microliters of extraction solution was added with a 200-microliter pipette, followed immediately by 25 microliters of tissue preparation solution. The solutions were mixed by briefly vortexing. After this, the samples were mashed up with a pipette tip until thoroughly masticated and incubated at room temp for 10 minutes. After this, I moved the samples to the heat block and incubated for a further 3 minutes at 95 C. After this, 100 microliters of neutralizing solution was added to the reaction to stop it, and it was placed on ice until needed for PCR.
After samples were prepared, a PCR reaction was prepared to amplify ambient sample DNA for identification of species. To begin, a 1:10 dilution was prepared by pipetting 18 microliters of DI H2O into a 1.5 mL tube labelled “1:10 JK1” and “1:10 JK2”. To this, 2 microliters of the DNA extraction performed above was added. After this, the tube was vortexed in a centrifuge to consolidate all liquid and mix the tube. After this, a master mix reaction was created as per the protocol in the handout, with enough made for 16 reactions based on the 13 samples in our group (102.4 microliters water, 160 microliters rx mix, 12.8 microliters each of forward and reverse primer extract). However, we were short 4 reaction volumes, and another member of our group had to use a different PCR master mix. I took 2 18 microliter aliquots of this master mix and added them to 2 separate PCR tubes. To these tubes, I added one 2 microliter aliquot of DNA extraction each. After the thermocycler was set up, I added my tubes, after which they were PCRed at the following parameters:
4 min of 94 initial denaturation
30 cycles of:
94 C for 30 sec (denaturing)
52 C for 40 sec (annealing)
72 C for 1 min (extension)
72 C for 10 min (final extension)
10C for hold
After this, professor John R. Paul collected the samples and ran aliquots of the DNA to enable a positive genetic ID for each fish species present in the sample.