Exonuclease Treatment on DNA Intended for Use in Electroporation

20240125 Preparing Samples and Treatment

  • Kept DNA on ice, flicked to mix and spun down
  • Want to do 1ug to 500ng for each sample
  • Prepared samples in 1.5mL tubes
sample DNA vol 10mM tris vol total DNA
20 12.9ul 17.1ul 1000ng
21 8.47ul 21.53ul 1000ng
22 12.6ul 17.4ul 1000ng
23 14.7ul 15.3ul 1000ng
24 11.9ng 18.1ng 500ng
25 9.4ul 20.6ul 500ng
  • Thawed NEBuffer 4 and ATP on ice, vortexed and spun down
  • Exo V enzyme kept on ice
  • To each tube added:
    • 4ul NEBuffer 4
    • 4ul ATP
    • 2ul exonuclease V
  • Tubes were flicked to mix and spun down
  • Placed tubes at 37C heat block for 1 hour
  • Warmed the oven to 70C because the other heat block was taken
  • After the 1 hour, the samples were transferred to a pre-warmed tube rack in the 70C oven for 30 min

20240125 Phenol Chloroform Cleanup

I want these samples after the cleanup to be more concentrated than the other times I have done this, so I decided to pool the samples into only 2 samples

  • Combined samples 20, 21, and 24 together into 1 1.5mL tube with clipped pipette tips
  • Combined samples 22, 23, and 25 together into 1 1.5mL tube with clipped pipette tips
  • Added 180ul of 10mM tris HCL to each tube to bring the volumes up to 300ul
  • Further processes were done in the hood
  • Added equal volume (~300ul) of cold phenol-chloroform isoamy alcohol
  • Inverted to mix
  • Placed tube on orbital shaker for 10 minutes
  • Centrifuged tube for 15 minutes at 16,000rcf at room temp
  • Removed the top layer into new final labeled tubes:
    • 20: 300ul
    • 22: 305ul
  • Added 0.1X volume of 3M sodium acetate to the tube
    • 20: 30ul
    • 22: 30.5ul
  • Added 2-2.5X volume of cold 100% ethanol to the tube: I added 750ul which is 2.5X
  • Mixed by inverting many times
  • Placed sample in the -20 overnight
  • Placed the centrifuge in the 4C to cool overnight

20240126 Finish Cleanup

  • Centrifuged tube at 4C for 30 minutes at 16,000rcf
    • I did see a pellet in both tubes
  • Pipetted off the supernatant into the waste
  • Added 500ul of cold fresh 80% ethanol
  • Inverted the tube twice
  • Centrifuged the tube at 4C for 30 minutes at 16,000rcf
  • Pipetted off the supernatant into the waste
  • Let the tube dry for ~15 minutes upside down
  • Resuspended the pellets in 20ul of 10mM tris
  • Let pellet resuspend for a few hours before qubiting
  • Qubit followed this protocol for high sensitivity reagents
    • 20: 28.8ng/ul
    • 22: 32.5ng/ul