A modified Alexander et al. tube protocol was followed for DNA extraction.
First, 5 1.5mL centrifuge tubes were labeled with a sample ID (PRK01, PRK02, PRK03, PRK04, PRK05). Then, 3 sterile 3.2 mm stainless steel beads were carefully added to each tube. About 6 small leaflets from each sample tube were added to the appropriate centrifuge tube that contained the beads. Forceps were cleaned between each tube to avoid cross contamination.
The centrifuge tubes were then placed in a modified reciprocating saw rack and the rack was then mounted to the saw. The tubes were reciprocated using this method for about 40 seconds, with the speed set at 3. This enabled the leaflets to be crushed into a fine powder.
The tubes were then centrifuged for about 15 seconds for plant dust to collect at the bottom.
Next, 430 µl of preheated grind buffer was added to each tube. Once added, the tubes were incubated at 65 °C for 10 minutes in a water bath. Tubes were mixed via inversion every 3 minutes during the 10-minute period.
130 µl of 3M potassium acetate (pH = 4.7) was added to each tube. The tubes were inverted several times to mix. Next, tubes were incubated on ice for 5 minutes. Following incubation, all centrifuge tubes were allowed to centrifuge at maximum force (14,000 rpm) for 20 minutes.
New 5 1.5mL centrifuge tubes were labeled with the sample IDs listed above. Supernatant (clear solution containing DNA) from the tubes that were centrifuged above was transferred into the appropriate newly labeled tubes. 1.5 volumes of binding buffer (contains guanidine hydrochloride – a hazardous chaotropic salt) were added to each tube containing the supernatant. 500µl of this mixture was transferred to the corresponding Epoch spin column tubes that were labeled with the appropriate sample ID.
Then, DNA bound to the silica membrane of each Epoch tubes was washed with 500 µl of 70% EtOH (EtOH was added to column) and was centrifuged at 15,000 rpm for 8 minutes (so that all liquid passed into collection tube). The liquid collected in the collection tube was discarded into an Erlenmeyer flask. This step was repeated.
Once the liquid in the collection tube was discarded for a second time, the Epoch tubes were centrifuged for 5 minutes at 15,000 rpm for 5 minutes. This was done to remove any additional ethanol.
The collection tubes were discarded and the columns were correspondingly placed into sterile, labeled 1.5 mL microcentrifuge tubes. Finally, 100 µl of preheated pure, sterile water was added to each column and the tubes were allowed to sit for 5 minutes. The tubes were centrifuged for 2 minutes at 15,000 rpm to elute the DNA. Tubes were placed in ice.