Wednesday, March 11, 2015

3 11 2015 DNAzol Isolation with Fresh Tissue

So we've been having trouble with our tissues from previous samplings. Since they have all been preserved dry in -20 conditions for months to over a year we decided to attempt an extraction on Olympia oysters fresh from a hatchery. I collected 4 oysters from a South Sound population from the Ken Chew Hatchery thanks to Puget Sound Restoration Fund and Ryan Crim. I performed the first half of the DNAzol isolation tonight with tissues that I dissected out of living animals. Samples ranged from 30-70 mg in size which is close enough to the proper DNAzol range. Like the last time I did a DNAzol extraction, I split the samples into spooled and remnants. Surprisingly, the DNA spooled very very well. There was a clear difference between how these samples behaved during the extraction process and the previous samples. All reagents and techniques are identical to the previous extraction so no difference should be observed unless there's something to fresh extractions.


  1. Dissected out grain of rice size piece of mantle tissue from each oyster and placed in collection tube. 
  2. Added 1 ml DNAzol reagent to tube. 
  3. Homogenized using pestle for 45 seconds
  4. Incubated at room temp for 10 minutes
  5. Centrifuged sample at 10,000 g for 10 minutes
  6. Extracted supernant and placed into a fresh tube
    1. Large goopy looking clumps in supernant that had clearly not compacted during centrifugation were present. 
  7. Added 500 ul 100% EtOH to supernant
    1. Instantly produced large amount of bright white material
    2. After inversion mixing, large goopy strings of precipitate formed on the bottom
  8. Spooled goopy material on the bottom of the tube and placed into new tube
  9. Centrifuged remaining supernant at 5,000 g for 5 minutes.
    1. No pellet formed in these tubes. 
    2. Slight film on tube walls.
  10. Added 1 ml 75% EtOH to tubes.
    1. In the original tubes nothing happened
    2. In the spool tubes bright white strings of material appeared and solution clouded up
  11. Incubated for 1 minute at room temp.
  12. Centrifuged tubes at 1,000 g for 2 minutes
    1. spooled tubes had large amount of material still in suspension
    2. Recentrifuged these tubes at 10,000 g for 2 minutes
    3. film and pellet formed from spooled tubes
  13. Removed EtOH as best I could
    1. Due to strings of material I couldn't remove all of it
  14. 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. 

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