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

Notes

  • Samples:
    • Pos-B: DiNV GS 2.1 FH 240hr Flask 7-18-2018
    • CC-B: DiNV GS 3 240hr CC Flask 7-18-2018
  • Using 600ul input this time to try to increase yield of DNA
  • Going to let the pellet dry for longer (30 min if possible) after the ethanol wash because in the regular extraction protocol they let their pellets dry out for a lot longer. Apparently ethanol can inhibit DNA from going into solution
  • Going to leave the samples resuspending for an extra day before trying the QC
  • Samples were left overnight in the fridge to avoid a freeze thaw cycle
  • Trying a new centrifuge this time for the precipitation that goes closer to 5000g

20211026

Washing Cells

  • Made fresh 70% ethanol and placed in the fridge
  • Placed 1X PBS and molec grade water on ice
  • Kept samples on ice
  • Set centrifuge on the 5th floor to go to 4 degrees
  • Inverted sample tubes before pipetting to mix up the cells
  • Clipped the ends of p1000 pipette tips for each sample
  • Transferred 600ul of each sample into their own 15mL tube
  • Added 9mL 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 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 to get the pellet to resuspend
  • Let tubes sit on ice for 10 minutes
  • Centrifuged 1,300rcf for 15 minutes at 4 degrees C
  • Removed supernatant without disturbing the pellet
    • There is a small pellet in each sample
  • Added 1mL cold buffer C1 to each tube
  • Added 3mL cold molecular grade water to each tube
  • Vortexed breifly
  • 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
    • I did see the pellet come up again and hopefully get fully mixed
  • 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
    • Note here that the tip for the POS-B sample drips a lot slower than the one for the CC-B sample, I had significant wait times with just that tip
  • Vortexed sample tubes briefly
  • Cut p1000 tips for each samples
  • Added total volume (~5mL) of each sample to their respective tips and let drip
  • Placed buffer QF in the incubator to warm to 50 degrees C
  • Added 7.5mL of buffer QC to each tip
  • Transferred tips to a new 15mL waste conical
  • Added 7.5mL of buffer QC to each tip
  • Transferred tips to new 15mL conicals labeled for final tubes
  • Added 5mL of warmed buffer QF to each tip

Precipitation

  • Added 3.5mL of 100% isopropanol to each elutent tube and inverted multiple times to mix
  • Centrifuged tubes in the Slusky Lab at 4 degrees C 4,816rcf for 30 min After this centrifugation, both conicals had completely cracked and broken. Sample liquid was not salvageable. Extraction attempt was stopped here. The tubes I had used were polystyrene not polypropylene which means they couldn’t stand very much force. I will need to try this extraction again with a new set of tubes, and I will have to ask Kent for more samples.