Third Test of the Hirt Method for Viral DNA Extraction With Phenol Chloroform Purification on DV-2 Cells Infected with DiNV, and p47 and RPL11 PCRs on Extracted DNA

Second extraction test did not go well because the DNA was too contaminated with something to get a PCR to work. I did a nanodrop on the extracts and the 260/280 ration was high (between 1.85 and 2.13) which indicates that the solution is basic. What I changed this time is that I centrifuged the samples twice after the chromosomal precipitation, I did not use phase lock tubes, I did the incubation for DNA precipitation in the -20 overnight, and I made new NaOAc.

New 3M NaOAc

  • Making 50mL
  • 12.3g sodium acetate
  • Dissolved in 35mL of molec grade water (took a while to dissolve, lots of mixing)
  • Checked pH with test strips, want it to be between 4.8 and 5.2
  • Original pH was between 6 and 7, way too high
  • I did many “titrations” of adding glacial acetic acid, but it came out to be about 1.2mL of glacial acetic acid added to get the pH to about 5 (test strips aren’t super accurate)
  • Then I increased the volume to 50ul with molec grade water

Lysis and Chromosomal DNA Precipitation

  • Placed small centrifuge in fridge at noon
  • Thawed 2 samples from Kent and a cell control Dv-1 sample to use as a sort of control for the extraction
  • Samples named by the volume used:
    • 1.5-2 : DiNV GS 20181123 Dv-1-2 day 10 lysate
    • 0.75 : DiNV GS 20181123 Dv-1-3 day 10 lysate
    • 0.5-C : DiNV GS 20181123 Dv-1-CC day 10 lysate
  • Mixed the tubes before transferring samples to 1.5mL tubes
  • Used clipped pipette tips to transfer volume to new 1.5mL tubes
  • Centrifuged tubes at 4C 1000rcf for 3 minutes
  • Each tube had a clear pellet
  • Removed supernatant from pellet
  • Added 1.5mL 1X PBS
  • Centrifuged tubes at 4C 1000rcf for 3 minutes
  • Removed supernatant from pellet
  • Resuspended pellet in 147ul 50mM Tris HCl, 10mM EDTA and 3ul of 5mg/mL RNase A
  • Added 150ul of 1.2% SDS to each tube
  • Inverted tubes, gently resuspended pellet with clipped pipette tips to mix and spun down
  • Incubated tubes on bench for 10 minutes to lyse all cells, waited until I couldn’t see any cell chunks anymore
  • 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
  • Immediately placed tubes on ice for 25 minutes incubation (this was increased from 15 minutes)
  • Centrifuged tubes for 15 minutes at 16,000rcf at 4 degrees C
  • Pipetted off supernatant into new 1.5mL tubes with clipped pipette tips
  • Centrifuged 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 ~475ul

Phenol Chloroform Extraction

  • Took tubes and setup to the hood
  • Added equal volume (475ul) of cold phenol-chlorofomr-isoamy-alcohol to the tubes
  • 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 was “perfect” in each tube
  • Transferred top aqueous phase to new final labeled tubes
    • I did not try to get absolutely everything, I did not want to suck up both phases
    • 400ul, 550ul, and 400ul were sucked up
  • Added 0.1X tube liquid volume (either 40ul or 55ul) of new 3M NaOAc to each tube
  • Added 900ul of cold 100% ethanol
  • Inverted tubes to mix
  • Placed tubes in the -20 overnight

DNA Precipitation 20220902

  • Centrifuged tubes at max speed at 4C for 30 minutes
    • Tubes had not frozen in the freezer
    • A small pellet was seen in all tubes after this
  • Pipetted off supernatant
  • Added 500ul of cold fresh 80% ethanol
  • Centrifuged tubes at max speed at 4C for 30 minutes
  • Removed all ethanol
  • Let tubes dry ~60 minutes upside-down on the bench
    • After drying I couldn’t see the pellet anymore…
  • Resuspended pellets in 25ul of molecular grade water and let sit on the bench for a few hours
  • Qubit that afternoon:
    • 1.5-2 : 63.7ng/ul
    • 0.75 : 35.5ng/ul
    • 0.5-C : 130ng/ul
  • The yield is nice here, but in theory the cell control sample shouldn’t have any DNA in this extraction because there isn’t any virus. So this is kind of sad. I expected that I could never get rid of all of the Dv-1 DNA but this is a lot.

p47 and RPL11 PCRs 20220906

  • p47
  • So I have 3 samples, plus 1 for a positive control, and plus 1 for a negative control
  • Made master mix on ice:
    • 5ul GoTaq * 5.5 = 27.5ul
    • 0.25ul p47_F * 5.5 = 1.375ul
    • 0.25ul p47_R * 5.5 = 1.375ul
    • 3.5ul molecular grade water * 5.5 = 19.25ul
  • Vortexed and spun down master mix
  • Added 9ul master mix to 4 strip tubes
  • Added 1ul of DNA sample to appropriate tubes
  • Added 1ul of 3mL HMW extraction to the positive control tube
  • Added 1ul of molecular grade water to the negative control tube
  • Vortexed and spun down strip tubes
  • Placed tubes in the p47 PCR program
  • RPL11
  • Using the same sample scheme as above
  • Made master mix on ice:
    • 5ul GoTaq * 5.5 = 27.5ul
    • 0.25ul p47_F * 5.5 = 1.375ul
    • 0.25ul p47_R * 5.5 = 1.375ul
    • 3.5ul molecular grade water * 5.5 = 19.25ul
  • Vortexed and spun down master mix
  • Added 9ul master mix to 4 strip tubes
  • Added 1ul of DNA sample to appropriate tubes
  • Added 1ul of 3mL HMW extraction to the positive control tube
  • Added 1ul of molecular grade water to the negative control tube
  • Vortexed and spun down strip tubes
  • Placed tubes in the RPL11 PCR program
  • Ran a 2% gel for 35 minutes at 90 volts after the PCR programs were done:

Clearly there is still Dv-1 DNA in all of these, I’m not sure what more I can do to minimize that…