This lab period utilized a modified version of Alexander et al. tube protocol for DNA extraction.
Materials and Methods
- 2.0 mL tubes
- Sterile 3.2 mm stainless steel beads
- Mimulus guttatus leaf tissue (preserved in silica)
- Tube rack
- Modified reciprocating saw rack
- p20 – p1000 pipets and pipet tips
- Grind buffer
- 3M pH 4.7 potassium acetate
- 1.5 mL tubes
- Binding buffer
- Epoch spin column tubes
- 70% EtOH
- Pure sterile water
I labelled 3 2.0 mL tubes with the codes RWCK 003 AJC, MAPL 005, and CATB – EVR. The first two samples were obtained by Alex, a graduate student working with Professor Paul. Three sterile 3.2 mm stainless steel beads were added to each tube and a small amount of leaf tissue from each sample was placed in their respective tubes. The tubes were placed in a rack, which then was placed in a modified reciprocating saw rack and was reciprocated on speed 3 for 40 seconds. The tubes were placed in the centrifuge for 18 seconds to pull plant dust down. 434 microliters of preheated grind buffer was added to each tube. The buffered grindate was incubated at 65 degrees Celsius for 13 minutes and 30 seconds and were mixed by inversion every three minutes. 130 microliters of 3M pH 4.7 potassium acetate was added and mixed by inversion. These were incubated on ice for 5 minutes and 5 seconds. Samples were then centrifuged at maximum force for 20 minutes. New 1.5 mL tubes were labelled with the aforementioned sample IDs and the supernatant was transferred to those tubes. Some precipitate was pipetted into sample 3. 1.5 volumes of binding buffer was added which was around 550 microliters for each of the samples. 650 microliters of the mixture was added to Epoch spin column tubes and centrifuged for 10 minutes at 15,000 rpm. The flow through was discarded in a hazardous waste container and then the process was repeated with another 650 microliters. The DNA bound to the silica membrane was washed by adding 500 microliters of 70% EtOH to the column and then centrifuged for 8 minutes. This step was repeated after discarding the flow through. The columns were then centrifuged for a final time for five minutes to remove residual ethanol. The collection tubes were discarded and the columns were placed in 1.5 mL microcentrifuge tubes that were labelled with the ID and date. 100 microliters of pure sterile H2O was added to each tube, which were allowed to stand for 5 minutes and then centrifuged for 2 minutes at 15,000rpm to elute DNA.
This lab period utilized Geneious, a program that performs various functions on DNA and protein sequence data, to analyze the data collected in Lab 02. Both the forward and reverse of EVR03 succeeded while EVR01, EVR02, and EVR04 did not return any usable data. To substitute, I am using the extra code BP03 which was reported as albacore tuna.
Forward and reverse primers of EVR03 and BP03 were obtained and put through Geneious and were assembled in their respective samples. A new folder was made and the documents were copied and de novo assembled. The sequences were trimmed at the ends where confidence was low and some corrections were made to ambiguous codes where there was a discrepancy between the base calls of forward and reverse primers. Consensus sequences were generated and the sequences were BLASTed and the top hits were compared against what the fish was advertised as. An alignment was built of the sequences with some of the hits–EVR03 was tested against 8 different hits while BP03 was tested against 9 different hits. The alignments were built with Multiple align and muscle alignment with default settings.
- For EVR03, it was listed as mackerel–the BLAST revealed that the sequence was in the Scombridae family and had a grade of 99.2% for both Thunneus obseus and Thunneus albacares, though I am inclined to believe it is T. obseus because it’s slightly higher than T. albacares in the BLAST details. For BP03, it was listed as albacore tuna. The BLAST revealed the sequence was closest to Thunnus alalunga, which is, in fact, the albacore tuna. This result had a grade of 99.7%.
- Polymorphisms in EVR03’s nucleotide alignments were found at sites 178 and 292. Ambiguous codes weren’t found in sites with polymorphisms. The first ten polymorphisms in BP03’s nucleotide alignments were found at sites 19, 31, 46, 277, 283, 301, 328, 352, 373, and 415. There were 18 polymorphic sites in all.
This class and lab period (12:45 to 17:00) was used as another field trip to collect samples of M. guttatus and observe them in their habitats. Two areas were visited: the Red Rock Beach Cold Spring and a lower trail path near Muir Woods.
The spring is located near Stinson Beach and potable water from the Mount Tamalpais watershed flows through three pipes located in the area. The constant flow of water means that even though the spring is located in a rather sunny and hot area (especially on the day of the lab where temperatures continued to climb well beyond a normal 70 degrees), M. guttatus is able to flourish there.
M. guttatus’s “landing strip” for bees is also pictured below.
Because it’s pollinated by bees and the area of M. guttatus is limited by its access to water, the populations of M. guttatus in the area are likely inbred due to that limitation.
The second area had much more access to water–even though the water level of the creek is lower due to the fact that we’re just getting into fall and the winter rains haven’t fallen yet, there’s still enough water to support various populations of M. guttatus and facilitating their gene flow through water and pollination.
Some populations were located in areas that could be flooded before they properly disperse their seeds, which introduces questions on effective population size vs. population size and how conservation efforts should approach species in different areas.