Trying High Molecular Weight Extraction of “Cell Control” (non-infected) Cell Culture Sample

Notes

  • Using Blood & Cell Culture DNA Midi Kit
  • Following recommendations from the Cell Culture samples in the Qiagen Genomic DNA Handbook
  • No centrifuge in the lab that holds 15mL tubes and is refrigerated, there is one on the 5th floor in a shared equipment room
  • Sample info: DiNV GS 2 240hr CC flask 7-18-2018

Prepared Beforehand

  • Thawed sample on ice (from -80)
  • 50mL conical of 1X PBS cold (put in fridge then on ice bucket before use)
  • 50mL conical of molecular grade water cold (put in fridge then on ice bucket before use)
  • 50ml conical of 100% isopropanol at room temp
  • 50mL conical of 70% ethanol cold (put in fridge then on ice bucket before use)
  • Buffer C1 is kept in the 4 degree fridge

Sample Prep

  • Snipped the end of a p1000 tip to make it “wide bore” to transfer the sample
  • Transferred sample to a 15mL tube (~1mL), the sample was in a cryovial so there was no way to spin it down before transferring
  • Added mL cold PBS to the 15mL tube and kept on ice
  • Made a balance 15mL tube with 10mL water, weighed to check if weights were within 0.2g difference
  • Went to the 5th floor shared equipment room (above McDonald lab) and set the centrifuge to 4 and 1,500rcf. Ran the centrifuge for 10 minutes to get the temp to cool down first
  • Centrifuged the 15mL tubes at 1,500rcf, 4 degrees C, for 10 minutes: after I could see a small pellet at the exact bottom of the tube
  • Took out the tube, placed on ice, and returned to 4055
  • Removed all supernatant without disturbing the pellet
  • Added another 10mL cold PBS
  • Took the tubes upstairs and centrifuged the 15mL tubes at 1,500rcf, 4 degrees C, for 10 minutes
  • Took out the tube, placed on ice, and returned to 4055
  • Removed all supernatant without disturbing the pellet
  • Added 2mL cold PBS
  • Added 3mL cold buffer C1
  • Added 6mL cold molecular grade water
  • Let the tube sit on ice for 10 minutes
    • After ~7 minutes I realized I hadn’t mixed the tube, so I inverted it a few times and made sure it sat for 15min total on ice before centrifuging
  • Took the tubes upstairs and centrifuged the 15mL tubes at 1,300rcf, 4 degrees C, for 15 minutes
  • Took out the tube, placed on ice, and returned to 4055
  • Removed all supernatant without disturbing the pellet
  • Added 1mL cold buffer C1
  • Added 3mL molecular grade water
  • Vortexed the tube briefly
  • Adjusted the balance tube to ~4mL
  • Set the oven in 4055 to 50 degrees C to warm up
  • Took the tubes upstairs and centrifuged the 15mL tubes at 1,300rcf, 4 degrees C, for 15 minutes
  • Took out the tube, placed on ice, and returned to 4055
  • Removed all supernatant without disturbing the pellet
  • Added 5mL buffer G2
  • Added 95mL Proteinase K (stored in the fridge)
  • Vortexed the sample for a few seconds
    • NOTE here I noticed the pellet unstick completely, I vortexed again and it disappeared. At this point I got concerned that the pellet did not get mixed in with the buffer C1, which was supposed to lyse cells
  • Put the conical in the 50 degree C incubator for 40 minutes

Genomic Tip Extraction

  • Set up the tip with a 50mL conical under it
  • While the samples were still incubating for ~10 min, added 5mL of buffer QBT to the tip and let it drip through
    • Finished in the 10 min
  • Took the sample out of the incubator: couldn’t see anything in the homogenate and it looked pretty clear
  • Added the 5mL of sample to the genomic tip and let it drip through
    • NOTE I forgot to clip the tip of the pipette tip this time, so this would be bad for the DNA
  • Dripping time: Did not let the sample finish dripping, it was not finished after 1 hour. It should not take more than 20 min max to go through the tip. Had to stop extraction because of time constraints/this is a sign it’s not working.

Thoughts for trying again

  • Use less volume of sample, try 1/3 of what is in the tube to 1. not use all of the sample, and 2. if there was too many cells this may help that. I am not sure if there were too many cells, my point of reference is what I used for tissue extractions, which was 60mg of tissue. Talking with Kent he said these samples have way less cells than that, which is why I used all of this sample
  • Vortex the sample well after the first addition of buffer C1, I don’t think inverting disrupted the pellet of cells at all. Buffer C1 is supposed to lyse cells and maybe the issue was that the cells weren’t lysed well
  • Another issue I ran into was the speed of the centrifuge upstairs. It doesn’t go any higher than 4,220rcf. Protocol says to precipitate the DNA at greater than 5000rcf. Looking it up, most protocols say to do 10,000rcf or more…
    • Options: try max speed on that centrifuge and see if it works
    • Put the 1.5mL 4055 centrifuge in the fridge, split up the eluted sample into 6 tubes and precipitate it that way
    • Ask around about a more capable temp controlled centrifuge