- Dissected out grain of rice size piece of mantle tissue from each oyster and placed in collection tube.
- Added 1 ml DNAzol reagent to tube.
- Homogenized using pestle for 45 seconds
- Incubated at room temp for 10 minutes
- Centrifuged sample at 10,000 g for 10 minutes
- Extracted supernant and placed into a fresh tube
- Large goopy looking clumps in supernant that had clearly not compacted during centrifugation were present.
- Added 500 ul 100% EtOH to supernant
- Instantly produced large amount of bright white material
- After inversion mixing, large goopy strings of precipitate formed on the bottom
- Spooled goopy material on the bottom of the tube and placed into new tube
- Centrifuged remaining supernant at 5,000 g for 5 minutes.
- No pellet formed in these tubes.
- Slight film on tube walls.
- Added 1 ml 75% EtOH to tubes.
- In the original tubes nothing happened
- In the spool tubes bright white strings of material appeared and solution clouded up
- Incubated for 1 minute at room temp.
- Centrifuged tubes at 1,000 g for 2 minutes
- spooled tubes had large amount of material still in suspension
- Recentrifuged these tubes at 10,000 g for 2 minutes
- film and pellet formed from spooled tubes
- Removed EtOH as best I could
- Due to strings of material I couldn't remove all of it
- Eluted samples with 300 ul of Nanopure water.
All in all, these samples behaved totally different. Previous samples would not spool and remained in supernant which formed a solid pellet. These samples spooled very well, leaving little in the supernant and formed a film and loose pellet of material. Adding Ethanol made DNA precipitate visibly unlike before.
I will run these samples on a gel tomorrow but I get the feeling they are going to have high quality DNA in them. If this works, future samples should be collected and isolated immediately or within a short time frame.