Genomic Tip Blood & Cell Culture DNA Midi Kit on One Cell Control and One DiNV Positive Cell Culture Samples

Notes

  • Following recommendations from the Cell Culture samples in the Qiagen Genomic DNA Handbook
  • For the isopropanol precipitation, I kept the samples in 1 tube and used the centrifuge on the 5th floor that only goes to 4,255rcf maximum speed. I was worried that there would be little DNA and that splitting it up into 6 tubes would mean nothing would pellet in any of them
  • Samples:
    • Pos: DiNV GS 2.1 FH 240hr Flask 7-18-2018
    • CC: DiNV GS 3 240hr CC Flask 7-18-2018

20211018

Washing Cells

  • Thawed samples on ice
  • Chilled 1X PBS, molecular grade water, and 70% ethanol on ice
  • Went to the 5th floor room and set the centrifuge to run for 15 min to chill it down and be prepared while I got the samples ready
  • Inverted samples when thawed, looked for chunks of cells to mix up
  • Clipped the ends of p1000 pipette tips for each sample
  • Transferred 300ul of each sample into their own 15mL tube
  • Added 10mL cold PBS to each tube
  • Centrifuged tubes at 1,500rcf for 10 minutes at 4 degrees C
  • Removed supernatant from each tube without disturbing the pellet
    • There was a visible very small pellet in each tube
  • Added 10mL cold PBS to each tube
  • Centrifuged tubes at 1,500rcf for 10 minutes at 4 degrees C
  • Removed supernatant
  • Added 2mL cold PBS to each tube
  • Added 2mL cold buffer C1 to each tube
  • Added 6mL cold molecular grade water to each tube
  • Vortexed tubes to get the pellet to resuspend: had to get it so the vortex would swirl all the way to the bottom of the tube for it to unstick
  • Let tubes sit on ice for 10 minutes
  • Centrifuged 1,300rcf for 15 minutes at 4 degrees C
  • Removed supernatant without distrubing the pellet
    • I could not really see a pellet now but I acted like there was one
  • Added 1mL cold buffer C1 to each tube
  • Added 3mL cold molecular grade water to each tube
  • Vortexed breifly
    • When vortexing there was a tiny white pellet that came up, good sign!
  • Centrifuged 1,300rcf for 15 minutes at 4 degrees C
  • Removed supernatant

Incubation

  • Added 5mL buffer G2 to each tube
  • Added 95mL proteinase K to each tube
  • Vortexed tubes briefly (here I did not see a pellet)
  • Placed tubes in the incubator at 50 degrees C for ~60 minutes

Genomic Tip Extraction

  • Set up two tips over 50mL conicals
  • Added 4mL buffer GBT to each tip and let drip (~5 min)
  • Vortexed sample tubes briefly
  • Cut p1000 tips for each samples
  • Added total volume (~5mL) of each sample to their respective tips and let drip (drip time 5-6min)
  • Placed buffer QF in the incubator to warm to 50 degrees C
  • Added 7.5mL of buffer QC to each tip (drip time ~8min)
  • Added another 7.5mL buffer QF to each tip (drip time 8-10min)
  • Transferred the tips to fresh 15mL conicals
  • Added 5mL warmed buffer QF to each tip (drip time ~6min)

Ethanol Precipitation

  • Added 3.5mL 100% isopropanol to each 15mL tube with the elution and inverted to mix
  • Centrifuged 15mL tubes in the centrifuge on the 5th floor for 30 min at 4 degrees C and the max speed: 4,255rcf
  • I did not see visible pellets after centrifugation
  • Kept tubes on ice when using at the bench
  • Removed supernatant from each tube and saved in a conical for later
  • Added 2mL of cold 70% ethanol to each 15mL tube
  • Vortexed tubes briefly
  • Centrifuged tubes for 10 minutes at 4 degrees C, 4,255rcf
  • Removed all supernatant, carefully using a p200 to get some of the liquid down near the “pellet”
  • Let the tubes air dry for ~15 minutes
  • Added 75ul of TE buffer to each tube
    • I saw something come up from the bottom of the tube when I added into the CC tube
  • Placed tubes in the incubator set to 55 degrees C for 1 hour
  • After 1 hour, placed the tubes on a rocking shaker pretty slow overnight

20211019

Qubit Quantification

  • Flicked to mix the tubes gently then spun them down (4012 centrifuge)
  • Warmed Qubit HS DNA assay for ~30 min out of the fridge
  • Measured each sample tube as “top” and “bottom” of the liquid because sometimes HMW DNA can be clumpy. Average should be the actual quantity
  • Pos top: 0.286ng/ul
  • Pos bottom: 0.477ng/ul
  • CC top: 0.401ng/ul
  • CC bottom: 0.508ng/ul
  • These quants are really low, with ~75ul total in each tube, this means there’s only about ~22ng total DNA in each. I don’t think with this concentration or amount it’s enough to run a gel on
  • I saved the first supernatant from the precipitation so I qubited that as well
  • Supernatant: 0.22ng/ul
  • This is a similar concentration as the actual samples, but the volume is way more! There is ~18mL of supernatant (both samples) meaning this should be 3.96ug of DNA total, or ~1.98ug per sample!
  • My guess is that 4,000rcf is not fast enough or 30 minutes is not long enough to precipitate the DNA efficiently. The handbook does say speeds faster than 5,000rcf are needed for efficient precipitation of DNA
  • There is also the chance that the isopropanol and the elution liquid in the supernatant are not compatable with the Qubit and this is not an accurate reading
  • Additional issues could be the small amount of sample I started with. The handbook recommends ~5 to 20 million cells for this extraction. I don’t think the 300ul I used had that much

Becuase I am limited on amount of sample currently, my next idea is to try to find a cold room I could put a big centrifuge in so I can get my precipitation spinning at ~10,000 rcf