Plant DNA Extraction

On Wednesday, October 11th, we extracted the DNA from Lupinus arboreus samples that we (and Dr. Paul) gathered earlier. We will do PCR with the extracted DNA in subsequent weeks, which will allow us to analyze how geographically distant populations are related.

Modified Alexander et al. tube protocol for DNA extraction

Procedure:

  1. First, I labeled five 1.5 mL centrifuge tubes with my sample’s label. I used “CRA”, as my samples came from Cowell Ranch Beach Access.
  2. I added three 3.2 mm stainless steel beads and a small amount of leaf tissue to each tube. I cleaned my tweezers with a ChemWipe between each use.
  3. We loaded tubes into a tube rack inside a modified reciprocating saw rack and mounted this to the saw. We then reciprocated the samples for 40 seconds.
  4. We spun our tubes in the centrifuge for 20 seconds to pull plant dust down from the lids.
  5. Next, I added 430 μL preheated grind buffer to each of my tubes.
  6. I incubated the tubes with grind buffer at 65° for 10 minutes. I mixed the tubes by inversion after 5 minutes.
  7. I added 130 μL pH 4.7 potassium acetate, inverted the tubes, and incubated them on ice for 5 minutes.
  8. I spun my samples in a centrifuge for 20 minutes at maximum force.
  9. I also labeled five new 1.5 mL tubes with the sample IDs. I transferred the supernatant from each centrifuged tube to these new tubes.
  10. I added 600 μL binding buffer to each of these tubes.
  11. Next, I added 650 μL of the mixture from step 10 to the Epoch spin column tubes.
  12. We centrifuged these tubes for 10 minutes at 15,000 rpm.
  13. I emptied the flow-through in each tube into a waste beaker.
  14. I then added the remaining volume from the 1.5 mL centrifuge tubes into the Epoch spin column tubes and centrifuged them for 10 minutes at 15,000 rpm. I emptied the flow-through into a waste beaker.
  15. I added 500 μL of 70% EtOH to the column, and I centrifuged the columns for 5 minutes to remove residual ethanol.
  16. I labeled 5 new 1.5 mL microcentrifuge tubes with my sample ID and date of extraction.
  17. I threw out the collection tubes in the biohazard waste, and I placed the columns in the microcentrifuge tubes.
  18. Finally, I added 100 μL preheated water (65 ° C) to each tube. I let the tubes stand for 5 minutes and then centrifuged for 2 minutes to elute the DNA.

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