Lab Entry 3: DNA Barcoding
Extraction of DNA:
Information was first taken to ensure that labels were correct between tubes and fish samples. Three varieties of fishes were collected and assigned the labels JW01, JW02, and JW03 for salmon, yellowtail, and tuna, respectfully. Gloves were used throughout the entirety of the procedure. Fish samples were derived as stated from a previous lab entry. The Sigma REDExtract-N-Amp Kit was also used for this lab which provided various components and solutions needed for extraction and amplification (next part).
Three 1.5-ml screw-cap centrifuges were labeled for each of the samples collected. Each of the samples were cut into small pieces (approx. 2-10 mg) on a plate using scalpels and forceps while cleaning the instruments between each sample with Ethanol in order to prevent cross contamination.
Using a p200 µl micro-pipette and unfiltered tips, 100 µl of Extraction Solution (ES) was added to each of the labeled tubes. 25 µl of Tissue Preparation Solution (TPS) was then added to each of the labeled tubes as well. To mix each of the solutions, the micro-pipette was pressed up and down. Solutions were derived from the kit mentioned earlier.
Each of the fish samples was then finally added to the corresponding microcentrifuge using forceps. Using hands, rather than the micro-pipette, a non-filtered pipette tip was used to gently mash the tissue sample. The sample was then left to incubate for 10 minutes at room temperature.
The samples were then moved to a heat block where they incubated at 95°C for 3 minutes which was measured using a timer. Samples were removed as close to the 3 minute mark as possible. Once removed from the heat block, 100 µl of Neutralizing Solutions (NS) was added using a p200 pipette and filtered tips and was then mixed by vortexing. The samples were then checked to make sure the labeling remained intact and then put on ice.
Amplifying COI from Fish:
For the amplification portion of the lab, the Sigma REDExtract-N-Amp Kit was used again. To amplify the genomic DNA (gDNA) that has been isolated from the previous part, PCR was conducted using Taq, dNTPs, and buffers provided from the kit.
DNA was first diluted 10-fold for optimal success when running PCR. A microcentrifuge tube was labeled “1:10” plus the name of the sample which also had my initials in the name. 18 µl of purified water was added to the tube followed by adding 2 µl of gDNA into the tube. The tube was gently flicked with finger to mix the solution.
A master mix was then made that contained all of the reagents being used for the solution when running the PCR that was shared among the lab table. In order to have the necessary amount of reagents for all of the table-mates, the master volume ratio was multiplied by 14 so that everyone would have enough solution. The master mix used had a total volume of 280 µl consisting of: 89.6 µl water, 140 µl REDExtract-N-Amp PCR rx mix, 11.2 µl forward primer, and 11.2 µl reverse primer. Extra reagents were added following the ratio for the purpose of ensuring that everyone would have enough solution for each of their gDNA samples.
For the three gDNA samples, three PCR tubes were labeled with corresponding labels on the top and sides of the tubes. 2 µl of 1:10 diltuion of gDNA was added to a PCR tube individually for each of the three samples. One table-mate created the negative control to be used during PCR that contained no gDNA in which case, the PCR tube remained empty for now. At this stage is when it was determined that a faulty pipette had been used when measurements were made. The pipette was incorrectly collecting solution amounts that may affect the results of PCR. The pipette was used earlier in the lab as well which may alter the outcome since ratios and amounts of reagents could have incorrectly been obtained. However, the master mix was not affected by the use of the faulty pipette and therefore, was used in the next step.
Once all of the tubes had their gDNA, 18 µl of the master mix was added into each of the PCR tubes, including the negative control tube. Pipette tips were changed between each of the samples. The samples were mixed via vortex and then placed on ice until all samples of lab-mates were ready for the next step.
All the PCR tubes including the negative control were then placed into the thermocycler and the reaction was started. The thermocycler takes approximately 1.5-2 hours and once completed, samples were placed into a freezer.
Settings for thermocycler:
- 94°C – 4 min (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)
- 10°C hold
The remainder of the lab will be conducted in the following lab session with a different lab post.