Using the Band 1, Band 2, and Pool of 1 and 2 Harvest Samples From DiNV Purification for Hirt DNA Extraction for Viral DNA

Based on the degraded DNA from the clarification pellet, I thought I would try extracting from what we think is purified virus with small amounts of fly DNA. I took 50ul sample from the purified band 1 (prefilter), 50ul from the purified band 2 (prefilter), and from the pool of harvest 1 and 2 supernatant (this has been kept at 4 C so expectation is that fly DNA is degraded here). Those are samples 7, 8, and 9 respectively. Sample information is here.

Lysis and Chromosomal DNA Precipitation

  • Placed small centrifuge in fridge
  • All processes were done in the fume hood
  • All samples were in 50ul liquid, so I added 100ul of 50mM Tris HCl, 10mM EDTA to each
  • Prepared 5mg/mL RNase A
    • 5ul molecular grade water
    • 5ul 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
    • It was hard to tell is anything happened here because all samples were basically a clear liquid to begin with, but I gently mixed a few times
  • 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
    • 7: 440ul
    • 8: 490ul
    • 9: 490ul

Phenol Chloroform Extraction

  • Still in the fume hood
  • Added equal volume (500ul) 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)
    • 7: 440ul
    • 8: 490ul
    • 9: 490ul
  • 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
  • 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
    • 7: 440ul
    • 8: 490ul
    • 9: 490ul
  • Added 0.1X tube liquid volume of new 3M NaOAc to each tube
    • 7: 44ul
    • 8: 49ul
    • 9: 49ul
  • 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

DNA Precipitation 20231028

  • Took tubes out of the freezer and centrifuged tubes at max speed at 4C for 30 minutes
    • Tubes had not frozen in the freezer
    • Maybe tube 9 had a pellet, all others looked like nothing
  • Pipetted off supernatant
  • Added 500ul of cold fresh 80% ethanol and inverted twice to mix
  • Centrifuged tubes at max speed at 4C for 30 minutes
  • Removed all ethanol
  • Let tubes dry ~60 minutes upside-down on the bench
  • Resuspended pellets in 25ul of 10mM tris HCl and let resuspend in the 4C until Monday

TPI and p47 PCRs and Gels 20231031

  • There are 3 samples
  • Include positive and negative control
  • Everything was thawed and kept on ice, and vortexed and spun down before use
  • TPI
    • 27.5ul GoTaq
    • 1.375ul TPI F
    • 1.375ul TPI R
    • 19.25ul molecular grade water
  • p47
    • 27.5ul GoTaq
    • 1.375ul p47 F
    • 1.375ul p47 R
    • 19.25ul molecular grade water
  • Vortexed and spun down mixes
  • Added 9ul of mixes to strip tubes
  • Added 1ul of diluted DNA to tubes
  • Added 1ul of water for the negative control and 1ul known virus positive DNA for the positive control
  • Vortexed and spun down strip tubes
  • Placed tubes in PCR programs, 30 cycles for each. PCR program information can be found here
  • Afterwards I did a broad range DNA qubit on each sample and they were all too low to read on with these reagents. This means each sample must be less than 2ng/ul concentration, which is far lower than the amount I’d need for electroporation…

All samples were run on a 1% gel : small rectangle 30mL 1X TAE and 0.3g agarose, .5ul of Midori stain