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Lab 8 entry- Modified Alexander et al. tube protocol for DNA extraction

  1. First, I labeled 3 2.0 mL tubes with my sample codes (KRS1-3 respectively).
  2. I added 3 sterile 3.2-mm stainless steel beads to each tube.
  3. Then, I added a small amount of leaf tissue into each tube, cleaning the tweezers between tubes to avoid contamination.
  4. Professor Paul loaded the tubes within a tube rack into the modified reciprocating saw rack and mounted the rack to the saw. He then turned it on speed 3 for 40 seconds.
  5. After, I centrifuged the tubes for 15-20 seconds at a fast speed to bring the plant dust down.
  6. I added 440 mL of preheated grind buffer to each tube.
  7. I incubated the buffered grandame at 65 degree C for 10 minutes in a water bath, mixing the tubes by inversion every 3 min.
  8. I then added 130 mL 3M pH 4.7 potassium acetate, inverted the tubes several times, and incubated the tubes on ice for 5 min.
  9. I centrifuged the tubes at maximum force for 20 min.
  10. I labeled new 1.5 mL tubes with the sample IDs and transferred the supernatant to the sterile tubes, avoiding transferring precipitate.
  11. Then, I added ~600mL of binding buffer to each tube, inverting to mix the solution.
  12. I added 650 mL of the new mixture to Epoch spin column tubes and centrifuged for 10 min, discarding the flow-through in a beaker.
  13. I repeated step 12 with the remaining solution.
  14. I washed the DNA bound to the silica membrane by adding 500 mL of 70% EtOH to the column and centrifuging at 15,000 rpm for 8 min until all liquid has passed through the membrane. I discarded the flow-through.
  15. I repeated step 14.
  16. Then, I centrifuged the empty columns at 15,000 rpm for an additional 5 min to remove any residual ethanol.
  17. I discarded the collection tubes and placed the columns in sterile, labeled 1.5 mL microcentrifuge tubes.
  18. Finally, I added 100 mL preheated pure sterile water to each tube and let it stand for 5 min before centrifuging for 2 minutes at 15,000 rpm. The solution left in the tube is the DNA.

kari

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