Using the Hirt Method and Exonuclease Treatment To Get DiNV DNA for Electroporation

Prep samples

  • 2 flasks of DiNV were infected on 1/30 with passage 4 DiNV
  • In the hood, ~5ul of medium was removed from each flask and frozen in a 15mL conical
  • The remaining fluid from the flasks was scraped and aliquoited into 6 tubes, each with ~800ul of fluid: 26, 27, 28, 29, 30, and 31
  • The samples were spun down at 2000g for 3 minutes
  • The supernatant was removed and put in the 15mL conical and frozen
  • The cell pellets proceeded with the extraction:

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
    • 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, 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
    • 26: 485ul
    • 27: 485ul
    • 28: 485ul
    • 29: 485ul
    • 30: 485ul
    • 31: 485ul

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)
    • 26: 485ul
    • 27: 485ul
    • 28: 485ul
    • 29: 485ul
    • 30: 485ul
    • 31: 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 )
    • I did not try to get absolutely everything, I did not want to suck up both phases
    • 26: 475ul
    • 27: 480ul
    • 28: 445ul
    • 29: 445ul
    • 30: 445ul
    • 31: 445ul
  • Added 0.1X tube liquid volume of new 3M NaOAc to each tube
    • 26: 47.5ul
    • 27: 48ul
    • 28: 44.5ul
    • 29: 44.5ul
    • 30: 44.5ul
    • 31: 44.5ul
  • 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
    • 26: 90.3ng/ul
    • 27: 105ng/ul
    • 28: 27.4ng/ul
    • 29: 119ng/ul
    • 30: 102ng/ul
    • 31: 284ng/ul

All sample information on extractions like these can be found here

20240303 Exonuclease Treatment

  • Kept DNA on ice, flicked to mix and spun down
  • Want to do 1ug for each sample if possible, sample 31 had so much DNA I did 3 sets from it
  • Prepared samples in 1.5mL tubes
sample DNA vol 10mM tris vol total DNA
26 11ul 19ul 1000ng
27 9.5ul 20.5ul 1000ng
28 14.5ul 15.5ul 400ng
29 8.4ul 21.6ul 1000ng
30 9.8ul 20.2ng 1000ng
31-A 3.5ul 26.5ul 1000ng
31-B 3.5ul 26.5ul 1000ng
31-C 3.5ul 26.5ul 1000ng
  • Thawed NEBuffer 4 and ATP on ice, vortexed and spun down
  • Exo V enzyme kept on ice
  • To each tube added:
    • 4ul NEBuffer 4
    • 4ul ATP
    • 2ul exonuclease V
  • Tubes were flicked to mix and spun down
  • Placed tubes at 37C heat block for 1 hour
  • Warmed other heat block to 70C
  • After the 1 hour, the samples were transferred to a pre-warmed tube rack in the 70C heat block for 30 min

Phenol Chloroform Cleanup

I want these samples after the cleanup to be more concentrated, so I decided to pool the samples into only 2 samples

  • Combined samples 26, 27, 28, and 29 together into 1 1.5mL tube with clipped pipette tips - sample 26-exo
  • Combined samples 30, 31-A, 31-B, and 31-C together into 1 1.5mL tube with clipped pipette tips - sample 30-exo
  • Added 140ul of 10mM tris HCL to each tube to bring the volumes up to 300ul
  • Further processes were done in the hood
  • Added equal volume (~300ul) of cold phenol-chloroform isoamy alcohol
  • Inverted to mix
  • Placed tube on orbital shaker for 10 minutes
  • Centrifuged tube for 15 minutes at 16,000rcf at room temp
  • Removed the top layer into new final labeled tubes:
    • 20: 305ul
    • 22: 305ul
  • Added 0.1X volume of 3M sodium acetate to the tube
    • 20: 30.5ul
    • 22: 30.5ul
  • Added 2-2.5X volume of cold 100% ethanol to the tube: I added 750ul which is 2.5X
  • Mixed by inverting many times
  • Placed sample in the -20 overnight
  • Placed the centrifuge in the 4C to cool overnight

20240304 Finish Cleanup

  • Centrifuged tube at 4C for 30 minutes at 16,000rcf
    • I did see a pellet in both tubes
  • Pipetted off the supernatant into the waste
  • Added 500ul of cold fresh 80% ethanol
  • Inverted the tube twice
  • Centrifuged the tube at 4C for 30 minutes at 16,000rcf
  • Pipetted off the supernatant into the waste
  • Let the tube dry for ~15 minutes upside down
  • Resuspended the pellets in 25ul of 10mM tris
  • Let pellet resuspend for a few hours before qubiting
  • Qubit followed this protocol for high sensitivity reagents
    • 20: 15.1ng/ul
    • 22: 9.17ng/ul

Again, all sample information on extractions like these can be found here