Hirt DNA Extraction of Dinn Cells Infected with DiNV for 2 Weeks to Prep DNA to use in Electroporation

20240110 Inoculating Flasks

  • In the cell culture hood and following sterile technique
  • 2 Flask of Dinn cells passage 11 (no mushroom extract) were made on 12/19 and allowed to grow
  • On the 10th they were inoculated with 90ul of passage 4 DiNV (vials 12-17 in this spreadsheet)
  • The flasks were rocked to distribute the fluid and left to incubate at 22C for 2 weeks

20240124 Harvesting Cells

  • In the cell culture hood and following sterile technique
  • Removed ~6mL from each flask and placed in a 15mL conical, this left about 1-2mL in each flask
  • Used a cell scraper to get up as much of the cells that I could in each flask
  • Aliquoited about 600-700ul of solution into 1.5mL tubes:
    • 20: ~600ul of flask C
    • 21: ~600ul of flask C
    • 22: ~600ul of flask C
    • 23: ~700ul of flask D
    • 24: ~700ul of flask D
    • 25: ~700ul of flask D
  • Kept these tubes on ice until use a few min later
  • The 15mL conical was frozen at -20

20240124 Hirt Extraction

*Lysis and Chromosomal DNA Precipitation

  • The small centrifuge was placed in the 4C at least an hour before use
  • All samples were spun down at 2,000g for 3 min
  • The supernatant was removed and put in the 15mL conical of supernatant from earlier and cell pellets were left
  • All processes were done in the fume hood
  • Added 147ul 50mM Tris HCl, 10mM EDTA to all the samples
  • Prepared 5mg/mL RNase A
    • 10ul molecular grade water
    • 10ul 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 ~20 minutes to lyse/digest all cells/virus
    • This seemed to work, the cell pellets had disappeared by 15 minutes, and I had 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
  • 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
  • Moved supernatant into new 1.5mL tubes
    • 20: 485ul
    • 21: 475ul
    • 22: 475ul
    • 23: 475ul
    • 24: 465ul
    • 25: 475ul

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)
    • 20: 485ul
    • 21: 475ul
    • 22: 475ul
    • 23: 475ul
    • 24: 465ul
    • 25: 475ul
  • 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 )
    • I did not try to get absolutely everything, I did not want to suck up both phases
    • 20: 480ul
    • 21: 455ul
    • 22: 460ul
    • 23: 460ul
    • 24: 455ul
    • 25: 460ul
  • Added 0.1X tube liquid volume of new 3M NaOAc to each tube
    • 20: 48ul
    • 21: 45.5ul
    • 22: 46ul
    • 23: 46ul
    • 24: 45.5ul
    • 25: 46ul
  • Added 1000ul of cold 100% ethanol to each tube
  • Inverted tubes to mix
  • Placed tubes in the -20 overnight

20240125 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
    • 20: 77.7ng/ul
    • 21: 118ng/ul
    • 22: 79.6ng/ul
    • 23: 68ng/ul
    • 24: 42.1ng/ul
    • 25: 53.6ng/ul

All sample information on extractions like these can be found here