Allyson Luber

10/22/2019

Modified Alexander et. al tube protocol for DNA Extraction

1. I started off with labeling 3 2.0 mL tubes with my sample code (001- MAPL, 003- RDRK, AEL-CATB)
2. Added three sterile 3.2-mm stainless steel beads to each tube provided by Professor Paul
3. Added a small amount of leaf tissue to each tube- we had to be careful to clean any tweezers or other tools used between tubes to avoid cross contamination
4. Afterwards, we loaded the tubes within a tube rack into the modified reciprocating saw rack and mounted the rack to the saw. Ours was done by Professor Paul for 40 seconds
5. Once done on the saw we briefly spun down the tubes in the centrifuge for 15-20 seconds to pull plant dust down from the lids
6. Next thing, we added 434 μL preheated grind buffer to each of our sample tubes
7. Incubated buffered grindate at 65 degrees celsius for 10 minutes in water bath, mixing the tubes by inversion every 3 minutes
8. Added 130 μL 3M pH 4.7 potassium acetate, whilst inverting tubes several times then incubating on ice for 5 minutes
9. Next, we spun the tubes in a centrifuge yet again at maximum force for 20 minutes, at 14,000 or 15,000 rpm for the tubes in a smaller microcentrifuge
10. Bought coffee in this 20 minute break (you’ll need it).
11. Labeled new 1.5 mL tubes with sample ID
12. When finished centrifuging, transferred supernatant to these sterile 1.5mL microcentrifuge tubes– MAKE SURE TO ONLY GET SUPERNATANT AND NO PRECIPITATE
13. Added 1.5 volumes binding buffer. It was around 600 μL of binding buffer, to be exact
14. Applied 650 μL of mixture from step 11 to special little tubies, called Epoch spin column tubes and centrifuged for 10 minutes (until all liquid has passed through) at 15,000 rpm in a centrifuge and discarded flow-through in an erlenmeyer flask
15. Repeat step 14 with the remaining volume from step 13
16. Washed the DNA bound to the silica membrane by adding 500 μL of 70% EtOH to the column and centrifuge at 15,000 rpm until all liquid has passed to the collection tube (8 minutes). Discard the flow-through
17. Repeat step 16
18. After discarding the flow-through from step 17, we centrifuged the columns for an additional 5 min to remove any residual ethanol
19. Discarded the collection tubes and placed the columns in sterile 1.5 mL microcentrifuge tubes– make sure these tubes are labeled with sample ID and date
20. Finally, we added 100 μL preheated (65 degrees celsius) pure sterile water to each tube. Let it stand for 5 minutes and then centrifuged for 2 minutes at 15k rpm to elute the DNA