Hirt Extractions of Dinn Cells and Concentrated Supernatant Infected with DiNV Round 1

Because the Hirt extractions of purified virus fluids were giving me very low yields, I decided to try using infected cells as my source of virus DNA.

20231107 Inoculating Flasks

I had 2 flasks of Dinn cells that had been growing for about 2 weeks and had gotten pretty confluent. I think these cells we ~passage 9 in my hands. These cells had been growing for the twoish weeks in medium without mushroom extract. I removed 3mL of medium and replaced it with 3mL of fresh medium, again without mushroom extract. I then gave each flask (A and B) 250ul of DiNV passage 4 fluid. This is the same fluid we have titered and is at ~238.75FFU/ul. I really wanted these cells to get super infected. I swished the flask gently back and forth to distribute the solution, then placed them in the 23C incubator until the next Tuesday.

20231114 Harvesting Cells and Starting Hirt Extraction

I decided I wanted to try a couple sample types, either the cell pellet from 1mL of cell and supernatant solution, the cell pellet from 500ul of cell and supernatant solution, or 50ul of concentrated supernatant. I had gotten 10kD MWCO tubes that were free from some other lab in the building and I thought I could concentrate down the supernatant to 50ul, which is a volume that works ok in the extraction procedure.

  • Used a cell scraper to free as much of the cells from the flask as possible
  • Aliquoited 1mL from each flask (A and B) into 1.5mL tubes
  • Aliquoited 500ul from each flask (A and B) into 1.5mL tubes
  • Aliquoted 1mL from each flask (A and B) into 1.5mL tubes to use for MWCO concentration
  • All of the other fluid/cells was aliquoted into 1mL incriments in 1.5mL tubes and frozen at -80, there were 5 per flask. Additionally I made 1 50ul samples for both flasks to use for a puregene extraction
  • The other samples were kept on ice
  • To concentrate the supernatant, I spun out the cells from the 2 tubes with 1mL each
  • Removed the supernatant into the MWCO tubes, which can hold 500ul each. So for flask A there were 2 MWCO tubes, and 2 for flask B
  • Those MWCO tubes were centrifuged at 4C for about 3 and a half hours at ~2000rpm. I only realized after that this should have been more like 1000g, and would have gone faster, but potentially slower is better on the virus
  • I had spun the samples down until they were about 50ul of solution left in them
  • I aliquoted these 50ul into new tubes to use for the extraction
  • All samples used in the extraction:
sample # sample type
10 1mL Dinn cells infected pellet rep A
11 1mL Dinn cells infected pellet rep B
12 500ul Dinn cells infected pellet rep A
13 500ul Dinn cells infected pellet rep B
14-A 500ul Dinn cell supernatant concentrated rep A
14-B 500ul Dinn cell supernatant concentrated rep A
15-A 500ul Dinn cell supernatant concentrated rep B
15-B 500ul Dinn cell supernatant concentrated rep B

All sample information can be found here

All samples were kept on ice until used in the Hirt extraction

20231114 Lysis and Chromosomal DNA Precipitation

  • The small centrifuge was already in the hood from concentrating the samples
  • All processes were done in the fume hood
  • For the samples still with supernatant (10-13), the cells were pelleted at ~2000g and the supernatant removed and discarded
  • Added 147ul 50mM Tris HCl, 10mM EDTA to samples 10-13
  • Added 100ul 50mM Tris HCl, 10mM EDTA to samples 14A, 14B, 15A, 15B
  • 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 ~15 minutes to lyse/digest all cells/virus
    • This seemed to work, the cell pellets had disappeared by 15 minutes
  • 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 with clipped pipette tips
  • 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
    • 10: 480ul
    • 11: 475ul
    • 12: 480ul
    • 13: 480ul
    • 14A: 460ul
    • 14B: 475ul
    • 15A: 475ul
    • 15B: 475ul

20231114 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)
    • 10: 480ul
    • 11: 475ul
    • 12: 480ul
    • 13: 480ul
    • 14A: 460ul
    • 14B: 475ul
    • 15A: 475ul
    • 15B: 475ul
    • Note that the 14-15 samples got very cloudy when I did this
  • 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 14-15 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: 480ul
    • 11: 480ul
    • 12: 480ul
    • 13: 485ul
    • 14A: 450ul
    • 14B: 380ul
    • 15A: 400ul
    • 15B: 400ul
  • Added 0.1X tube liquid volume of new 3M NaOAc to each tube
    • 10: 48ul
    • 11: 48ul
    • 12: 48ul
    • 13: 48.5ul
    • 14A: 45ul
    • 14B: 38ul
    • 15A: 40ul
    • 15B: 40ul
  • 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

20231115 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, but some were very hard to see
  • 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 let resuspend at room temp overnight