Using the Hirt Method To Extract DiNV DNA for Later Comparison with Puregene Extraction

Using samples 36, 37, 38, and 39 that were already cell pellets frozen at -80, find info here

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 100ul 50mM Tris HCl, 10mM EDTA to all the samples
  • Added 4ul of 4mg/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 ~20 minutes to lyse/digest all cells/virus
    • This seemed to work, periodically inverted the samples throughout that time
  • 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 (I did not do a second centrifuge because it always seemed unnecessary)
  • During this time I made fresh 80% and 100% ethanol and put them in the -20 freezer to cool
  • Moved supernatant into new 1.5mL tubes
    • 36: 475ul
    • 37: 485ul
    • 38: 457ul
    • 39: 485ul

Phenol Chloroform Extraction

  • Still in the fume hood
  • Added equal volume of cold phenol-chloroform-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)
    • 36: 475ul
    • 37: 485ul
    • 38: 457ul
    • 39: 485ul
  • Mixed tubes up and down and then put them on an orbital mixer in the hood for 10 minutes, the samples did become cloudy at this time
  • 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, there was a small white layer between the two phases
  • Transferred top aqueous phase to new final labeled tubes (did not use clipped tips here for better pipetting )
    • 36: 440ul
    • 37: 430ul
    • 38: 400ul
    • 39: 427ul
  • Added 0.1X tube liquid volume of new 3M NaOAc to each tube
    • 36: 44ul
    • 37: 43ul
    • 38: 40ul
    • 39: 42.7ul
  • Added 1000ul of cold 100% ethanol to each tube
  • Inverted tubes to mix
  • Placed tubes in the -20 overnight

20240303 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 that was pretty visible
  • Pipetted 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
  • Pipetted off supernatant into a waste container
  • Let tubes dry ~30 minutes upside-down on the bench
  • Resuspended pellets in 20ul of 10mM tris HCl and left on the bench a few hours before qubiting
  • Qubit followed this protocol for broad range reagents
    • 36: 160ng/ul
    • 37: 203ng/ul
    • 38: 237ng/ul
    • 39: 160ng/ul