Another Set of Hirt DNA Extractions for DiNV DNA using Pellet of Cells Infected with DiNV and the Clarification Pellet from DiNV Purification

I had some leftover pellets of cells infected with DiNV from a previous hirt extraction where I only used the supernatant. Those 2 samples I called 16 and 17. Additionally I took two more samples of the clarification pellet from DiNV purification to use for the extraction. That pellet had been frozen at -80C by Kent, so it did have to go through a freeze thaw to get used. Those samples were 18 and 19. These probably aren’t the best samples (a frozen and thawed pellet and the clarification pellet which I know has degraded DNA) but I want to use this DNA to test and exonuclease on to see if that gets rid of non-circular DNA.

For the clarification pellet samples I added 100ul to 1.5mL tubes, spun them down at 2000rpm for 1 minute and removed the supernatant. It turned out that the cell pellet samples still had some liquid in them but I decided not to remove it.

Lysis and Chromosomal DNA Precipitation

  • The small centrifuge was placed in the 4C at least an hour before use
  • All processes were done in the fume hood
  • Added 147ul 50mM Tris HCl, 10mM EDTA to all the samples
  • Prepared 5mg/mL RNase A
    • 6ul molecular grade water
    • 6ul 10mg/mL RNase A
    • Vortexed and spun down to mix
  • Added 3ul of 5mg/mL RNase A to each sample
  • Added 150ul of 1.2% SDS to each tube
  • Inverted tubes, gently resuspended pellet with clipped pipette tips to mix
  • Incubated tubes on bench for ~25 minutes to lyse/digest all cells/virus
    • This seemed to work, the cell pellets had disappeared by 15 minutes, the clarification pellet samples got better, but I don’t think everything got broken down and I might have added too much sample
  • Precipitated chromosomal DNA and cellular debris by added 210ul of 3M CsCl, 1M potassium acetate, 0.67M acetic acid
    • A white precipitate immediately started forming in the tubes, I pipette mixed the solution with clipped pipette tips
  • Immediately placed tubes on ice for 30 minutes incubation
  • Centrifuged tubes for 15 minutes at 16,000rcf at 4 degrees C
  • During this time I made fresh 80% and 100% ethanol and put them in the -20 freezer to cool
  • Pipetted off supernatant into new 1.5mL tubes
  • Centrifuged new tubes for 15 minutes at 16,000rcf at 4 degrees C
    • There was basically no new precipitate here that pelleted, but samples 18 and 19 did have floating debris that I could not get avoid picking up
  • Moved supernatant into new 1.5mL tubes
    • 16: 490ul
    • 17: 500ul
    • 18: 500ul
    • 19: 500ul

Phenol Chloroform Extraction

  • Still in the fume hood
  • Added equal volume of cold phenol-chlorofomr-isoamy-alcohol to the tubes (Note!! phenol-chloroform-isoamy-alcohol is in two phases, I think you are supposed to use the bottom layer for this, see x)
    • 16: 490ul
    • 17: 500ul
    • 18: 500ul
    • 19: 500ul
  • Mixed tubes up and down and then put them on an orbital mixer in the hood for 10 minutes
  • Centrifuged tubes at 16,000rcf at room temp for 15 minutes
  • Looked for phase separation
    • Phase separation looked great, there were two distinct layers in each tube, however in the 18-19 tubes there was a white film in between the two layers, which ended up making it hard to remove the top layer below
  • Transferred top aqueous phase to new final labeled tubes (did not use clipped tips here for better pipetting )
    • I did not try to get absolutely everything, I did not want to suck up both phases
    • 10: 490ul
    • 11: 500ul
    • 12: 480ul
    • 13: 470ul
  • Added 0.1X tube liquid volume of new 3M NaOAc to each tube
    • 10: 49ul
    • 11: 50ul
    • 12: 48ul
    • 13: 47ul
  • Added 900ul of cold 100% ethanol to each tube
  • Inverted tubes to mix
    • I did not see anything that looked like DNA
  • Placed tubes in the -20 overnight

20240105 DNA Precipitation

  • Took tubes out of the freezer and centrifuged tubes at max speed at 4C for 30 minutes
    • Tubes had not frozen in the freezer
    • Each tube looked like they had a pellet
  • Poured off supernatant into a waste container
  • Added 500ul of cold fresh 80% ethanol and inverted twice to mix
  • Centrifuged tubes at max speed at 4C for 30 minutes
  • Poured off supernatant into a waste container
  • Let tubes dry ~30 minutes upside-down on the bench
  • Resuspended pellets in 25ul of 10mM tris HCl and put in the 4C