Test eDNA Extractions More Samples
4 seawater samples were filtered according to the previous protocol
Samples were separated into 4 different test regimes. Pelleted means centrifugation after digestion and discarding the pellet produced.
| Sample | Incubation Time | Pelleted? |
|---|---|---|
| 1 | overnight | no |
| 2 | overnight | yes |
| 3 | 6 hours | no |
| 4 | 6 hours | yes |
DNA Extraction
DNA was extracted from both samples with the Qiagen DNeasy Kit and modifications from Renshaw et. al
- Before extraction, the benchtop was wiped down with 10% bleach, DI water, then 70% ethanol
- Added to each tube with the frozen filters:
- 567µl buffer ATL
- 63µl Proteinase K
- Two samples were digested at 65 degrees C for overnight on the thermomixer and shaking at 600rpm
- Two samples were digested at 65 degrees C for 6 hours on the thermomixer and shaking at 600rpm
- After incubation, the filters were taken out of the 1.5mL tubes to get at the liquid with cleaned forceps (same as above). Excess liquid was squeezed out as best as possible with the forceps into new 1.5mL tubes
- Liquid from one original 1.5mL tube from each extraction time was centrifuged briefly before transferring the supernatant to new 5mL tubes. On the other 2 tubes the entire sample was transferred to 5mL tubes.
- Added to each 5mL tube and then vortexed
- 630µl buffer AL
- 630µl 100% EtOH
- The sample had to run through the spin column 3 times.
- Added 600µl sample to the spin column then centrigued at 8000rpm for 1 minute
- Transfer column to new collection tube
- Repeat above steps twice more to run all sample though the column
- Added 500µl buffer AW1 to the columns then centrifuged at 8000rpm for 1 minute
- Transferred columns to new collection tubes, added 500µl buffer AW2 and centrifuged at 14000rpm for 3 minutes
- Transferred columns to final 1.5mL tubes
- Added 50µl warmed (56 degrees C) buffer AE directly to the filter
- Incubated at room temperature for 5 minutes
- Centrifuged at 8000rpm for 1 minute
- in the same 1.5mL tube repeated above steps with 30µl warmed buffer AE
- Extracted DNA was frozen at -20 and a gel was run the next day
- A 2% agarose gel was made with GelRed and run for 60 minutes
