October 17, 2018

Using molecular markers to obtain a greater understanding of the degree of population differentiation and gene flow between populations of mimulus cardinalis, this phase of the experiment focused on the extraction of DNA from plant tissue. Mimulus cardinalis from various areas were collected and preserved in test tubes using silica, which quickly dried the plant tissue to enable better DNA preservation. Three samples of Mimulus cardinalis were provided in addition to their sample identification codes: JP1292, JP1304, and JP1302. DNA was extracted from these samples according to the following procedure:

1. The species name, sample IDs, and location (when available) was recorded on a chart, such as the one shown below.
   

   Sample ID	Location	Species
   JP1292		Mimulus cardinalis
   JP1304		Mimulus cardinalis
   JP1302		Mimulus cardinalis

2. The IDs were used to label three 2.0-mL tubes with a sharpie pen on the tube’s side and cap.
3. Three sterile, stainless, 3.2-mm metallic beads were added to each tube.
4. Taking the tubes containing the samples, a small portion of dried leaf tissue was broken off (approximately a fingertip’s amount) and placed into the 2.0-mL labeled tube. To avoid contamination, the tweezers were cleaned and wiped after transference of each sample. Repeat with all three samples.
5. The tubes were lined on a tube rack along the middle section and mounted onto a modified reciprocating saw rack.
6. Ensuring that the blade was secured and locked into the saw, the plug was inserted into the outlet and the saw was powered on and left for 40 seconds on speed 3.
7. The tubes were centrifuged for 15-20 seconds — making sure the centrifuge was balanced — to bring the powdery plant dust down from the tube caps.
8. 434-µL of preheated grind buffer was pipetted into each tube, switching filtered tips after each sample to avoid possible contamination events.
9. The tubes were fixed onto floaters inside a 65°C water bath for ten minutes, where they were inverted every three minutes to mix tube contents.
10. 130-µL of 3M, pH 4.7 potassium acetate was added into each tube, switching filtered tips after each sample. Tubes were inverted several times to induce mixing then placed on ice for five minutes.
11. Afterwards, centrifuge tubes for 20 minutes.
12. New 1.5-mL tubes were labeled with IDs.
13. Tubes were retrieved from the centrifuge, displaying stark contrast between supernatant and pellet/precipitate. The supernatant was transferred into the newly labeled 1.5-mL tubes corresponding to each sample, taking care to avoid pipetting precipitate.
14. After recording the approximate amount of supernatant obtained and comparing with the desired end volume of solution, 600-µL of binding buffer was added to each sample to reach an approximate final volume of 1.5-mL in each sample. Filtered tips were changed after transference of each sample.
15. 650-µL of the solution within each 1.5-mL tubes was pipetted into the Epoch spin column tubes, switching tips in between, then centrifuged for ten minutes.
16. The liquid that flowed through was discarded, and the remaining volume of solution within the 1.5-mL tubes were pipetted into the corresponding column tubes and centrifuged, making sure to discard the liquid flow-through once again.
17. 500-µL of 70% EtOH was added to each column tube and centrifuged for eight minutes to allow the liquid to pass through and wash the DNA bound to the silica membrane. The liquid that passed through was discarded. Repeated this washing step with EtOH twice.
18. Placed tubes in centrifuge for another five minutes to remove residual EtOH.
19. Labeled new 1.5-mL microcentrifuge tubes with IDs and date, then placed the columns (blue tubes, top part) into these sterile tubes while discarding the collection tubes (transparent tubes, bottom part).
20. 100-µL of preheated pure H2O was added to each tube through columns, left alone for five minutes, then centrifuged for two minutes to elute the DNA. Columns were then discarded and the microcentrifuge tubes holding extracted plant DNA were relinquished to the instructor.

Some difficulties occurred with the operation of the centrifuge, though this occurrence did not affect the extraction steps, and only slightly prolonged the length of this experimental phase. The extracted DNA solutions appeared to be of varying shades of brown-green despite belonging to the same species, which may possibly be attributed to the difference in the populations’ environments and habitats.