Using the Puregene Cell and Tissue Kit to Extract DNA from the DiNV Positive and Cell Control Samples to Get a Baseline of Extraction Efficiency

Notes

  • Following a mix of the bacterial and the 15 flies protocols from the lab drive because my samples are in liquid from
  • After looking at the sample volumes, I decided to try 300ul again to match the previous HMW extraction, and it looked like I had a little over 1mL left in the samples so I thought that volume would still leave me with enough volume for another HMW extraction attempt
  • Samples were kept in the fridge overnight (plan to do HMW extraction the next day) to avoid another freeze-thaw cycle
  • Samples still are:
    • Pos: DiNV GS 2.1 FH 240hr Flask 7-18-2018
    • CC: DiNV GS 3 240hr CC Flask 7-18-2018

Steps

  • Thawed samples on ice
  • Transferred 300ul of each sample to new 1.5mL tubes
  • Centrifuged tubes for 30 seconds at 13,000 rpm
  • Visible cell pellet after this
  • Removed the supernatant
  • Added 300ul cell lysis solution to each sample and pipetted to mix/break up the pellet
  • Made diluted RNase A (need 4mg/mL)
    • 24ul molec grade water
    • 1ul 100mg/mL RNase A
  • Added 1.5ul diluted RNase A
  • Vortexed and spun down samples
  • Incubated samples at 37 degrees C for 40 minutes
  • After incubation, placed tubes on ice for 1 minute to chill
  • Added 100ul of protein precipitation solution to each tube
  • Vortexed tubes for 10 seconds
  • Placed tubes on ice for 5 minutes (tubes got cloudy)
  • Centrifuged tubes for 3 minutes at 14,000rpm
  • Visible white pellet after this
  • Made 2 new tubes with 300ul of isopropanol in each
  • Moved ~330ul of supernatant to the new isopropanol tubes
  • Inverted tubes ~50 times to mix
  • Centrifuged tubes at 14,000rpm for 5 minutes
  • Not really able to see a pellet after this
  • Removed supernatant as best as possible
  • Added 300ul of 70% ethanol to each tube
  • Inverted tubes to wash
  • Centrifuged tubes at 14,000rpm for 1 minute
  • Again hard to see if there was a pellet
  • Removed supernatant as best as possible
  • Inverted tubes on a kimwipe for 5 minutes to dry
  • Added 25ul of DNA hydration solution to each tube
  • Pipette mixed and let DNA sit overnight on the bench to resuspend

Qubit 20211026

  • NOTE: written protocol is incorrect: should have let the pellets dry from ethanol for at least half an hour. My samples may have had poor resuspension because of left over ethanol
  • I might have to re-dry my samples then resuspend them to get a better idea of the quant
  • This also means I should probably increase the drying time of the HMW extraction after the ethanol wash because of the same issue
  • Qubit anyways to see what I have:
    • POS: 21.6ng/ul
    • CC: 3.44ng/ul
  • With 25 total ul, this means I got 540ng and 86ng total DNA respectively. This is more than with the HMW extraction and with the same amount of input. So I should have gotten more DNA with that. However this yield is still less than what should be expected based off of the estimates of numbers of cells in these volumes