Processed

GWC01

GWC02

GWC03

GWC04

GWC05

From Grey Whale Beach Site 3

Protocol:

1. Labeled 1.5 ml centrifuge tubes with correct sample
2. Added 3 sterile 3.2-mm stainless steel beads to each tube
3. Added small amount of leaf tissue to each tube, wiped between
4. Loaded tubes within a tube rack into the modified reciprocating saw rack and mounted rack into saw
5. spun tubes down about 20 seconds on fast speed
6. added 434 microliters of preheated grind buffer to each tube
7. incubate buffered grandame at 65 degrees celsius for 10 min in water bath, mixing tubes by inversion every 3 mins
8. add 130 microliters of 3M pH 4.7 potassium acetate, invert tubes several times and incubate on ice for 5 min
9. Spin in a centrifuge at maximum force (about 14000-15000 rams) for 20 min
10. Label new 1.5 ml tubes with sample ID
11. Add 1.5 volumes binding buffer
12. Add 650 microliters of mixture from step 11 to epoch spin column tubes and centrifuge for 10 min at 15,000 rpm in a centrifuge and discard flow-through
13. repeat step 12 with the remaining volume from step 11
14. wash with 500 ml of 70% EtOH then centrifuge at 15000 rpm until all liquid has passed through. discard flow-through
15. repeat 14
16. centrifuge columns at 15000 rpm for 5 more mins
17. discard tube then place columns in 1.5 ml labeled micro centrifuge tube
18. add 100 microliters preheated sterile h20 to each tube. let stand for 5 mins then centrifuge for 2 min at 15000 rpm to elute DNA