Lab 8: DNA Extraction

We extracted DNA from samples of Mimulus cardinalis and Mimulus guttatus. Most samples were from M. cardinalis centered around collections from Mt. Tam, but also including collections from Mt. Diablo, Big Sur, and near Mono Lake.

My samples were JP1308 (M. cardinalis), and JP1316 and JP1320 (both M. guttatus from Mill Creek near Mono Lake).

We used a modified Alexander et al protocol. Steps are as follows, including notes and details about my extraction today:

  1. Add 3 sterile 3.2 mm stainless steel beads to labeled 2.0 mL tubes.
  2. Add leaf tissue about the size of a thumbnail to tube. Clean tweezers to avoid contamination.
  3. Load tubes onto reciprocating saw. Run at speed 3 for 40 sec. Check if samples were broken up properly. The samples from my table broke up well.
  4. Spin down tubes for 20 seconds to pull plant dust down.
  5. Add 434 µL heated grind buffer. (note: I added 440 µL because the p1000 was not precise enough to do 434 µL)
  6. Incubate samples with buffer at 65 °C for 10 min in water bath. Invert tubes to mix every 3 minutes. (Note: incubation can be slightly longer than 10 min, but should not be less)
  7. Add 130 µL 3M pH 4.7 potassium acetate. Invert tubes several times to mix and incubate on ice for 5 minutes. (Note: again incubation can be longer than 5 minutes but shouldn’t be less)
  8. Spin on centrifuge at max speed (15,000 rpm) for 20 minutes. This pulls down solid leaf material so that we can take off the supernatant, which contains DNA.
  9. Into new, labeled 1.5 mL tubes, transfer supernatant by carefully pipetting to avoiding up-taking any precipitate. This ended up being about 400µL for all 3 of my samples.
  10. Add binding buffer to supernatant at 1.5x the volume of supernatant retrieved in the last step. For me, this was 600µL for each of my samples (400*1.5=600). (Note: binding buffer contains hazardous material so dispose of it properly)
  11. Add 650 µL of mixture from previous step to Epoch spin column and centrifuge for 10 min at 15,000 rpm. The spin column contains a silica filter that will bind to DNA under certain conditions. These tubes have a max volume of ~700µL, so we fill them up a little below that. When we spin the solution down, the silica gel allows the DNA to bind while letting the rest of the solution flow through. After centrifuge, discard flow-through into hazardous waste.
  12. Repeat step 11 with any leftover supernatant-binding buffer mixture. (Note: I noticed a small piece of leaf tissue in sample JP 1320)
  13. Add 500µL 70% ethanol to spin column and centrifuge at 15,000 rpm for 8 min. This step washes the bound DNA with ethanol, removing anything that may have bound to the filter that we do not want in our final extraction. Discard flow-through.
  14. Repeat step 13.
  15. Centrifuge columns at 15,000 rpm for 5 min without adding anything to the spin column. This helps pull through or dry out any ethanol still in the silica filter.
  16. Move spin columns into new, labeled 2.5 mL tubes. Add 100µL 65°C pure H2O to each spin column. Let stand for 5 min to allow water to soak into filter, then centrifuge for 2 min at 15,000 rpm to elute DNA.
  17. Discard spin column, and save flow through because your DNA is in here.

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