Monday, March 2, 2015

3 2 2015 DNAzol Extraction Attempt 2

A little over a week ago I attempted to isolate DNA using DNAzol and DNEasy to collect high molecular weight DNA. You can see the results here. I originally thought we had collected HMW DNA as the smears were mostly above the 1000 bp portion of the ladder. After discussing the results with Brent and Steven, it appears the HMW DNA is not large enough and should be a tightly condensed band at the top. Today I am extracting more DNA using DNAzol to try to extract only the HMW DNA using a spooling technique. The samples I'm using are the same ones from before since I had plenty of left over tissue (9-19-2014 1N9-12 18,19,20,21). To compare the spooling technique to the centrifuge technique, I spooled as much DNA as I could and transferred it to a clean second tube. Then centrifuged the original sample tube to collect a pellet. If I successfully collected the HMW DNA with the spooling it shouldn't appear in the centrifuged sample. If I didn't collected it, then the spooled sample should have little to no DNA. The rest of the protocol is as follows. I will run a gel on the samples this afternoon to check the sample quality.

DNA Isolation:

  1. Subsampled very small portion of tissue from original tubes and placed into homogenization tube. 
  2. Added 1 ml DNAzol to each tube
  3. Homogenized with 10 strokes of the pestle.
  4. Incubated at room temp for 10 minutes.
  5. Centrifuged at 10,000 g for 10 minutes.
  6. Transferred supernant to new tube.
  7. Added 0.5 ml 100% EtOH. 
    1. Originally saw a milky precipitate form but then disappear after mixing through inversion.
  8. Mixed through inversion (8 inversions)
  9. Incubated at room temp for 3 minutes. 
  10. Stuck a clean 200 ul pipette tip into solution and gently swirled solution around tip to spool DNA. Transferred to new labelled 1.5 ml tube and gently slid the tip across the inner surface of the tube.
    1. No visible spooled DNA. I repeated spooling multiple times to collect as much as possible but saw no visible DNA.
  11. Added 1 ml 75% EtOH to spooled DNA tube.
  12. Centrifuged original tube 5,000 g for 5 minutes. 
  13. Removed supernant from centrifuged tube.
  14. Added 1 ml 75% EtOH to Centrifuged DNA tube.
  15. Centrifuged all tubes at 1,000 g for 2 minutes to collect DNA in the bottom. 
  16. Carefully removed all remaining EtOH from all tubes. 
  17. Added 300 ul Nanopure water to each tube.
  18. Mixed through pipetting. 
  19. Stored at 4C until I can run the gel. 
I completed the gel run this evening for quality checking. It appears the spooling failed but upon examination of the centrifuged samples that there is a load of HMW DNA remaining in the well that did not travel through the gel. So the DNAzol works with the centrifuge extraction. 

0.8% Agarose Gel with Low TAE and 5 ul of EtBr. Ran gel at 120 v for 40 minutes.
Gel Layout

SampleLadder1N9-12 181N9-12 191N9-12 201N9-12 211N9-12 181N9-12 191N9-12 201N9-12 21LadderEmptyEmpty
Full Gel

High Molecular Weight DNA remaining in Well.

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