Second Exonuclease V Treatment Test on Hirt Extracts to Attemp to Remove Linear DNA and 6 Hour Gel

Because the gel of the previous treatment test was really hard to see for one of the crucial samples, I decided to try the treatment and cleanups again on just the cell pellet samples. Sample information can be found here

20240118 Exonuclease Treatment

  • Followed exact same process as the first use, where the protocol was based off of this protocol
  • I used the rest of the DNA in the 16 and 17 samples to try to maximize what I could
sample DNA 10mM Tris to 30ul total DNA
16 16ul 14ul ~900ng
17 16ul 14ul ~900ng
  • All DNA was kept on ice, flicked to mix, and spun down before use
  • All reagents were thawed on ice, vortexed to mix, and spun down before use
  • The enzyme was not frozen, and was spun down before use
  • DNA was diluted into 1.5mL tubes
  • To each diluted sample tube:
    • Added 4ul of NEBuffer 4
    • Added 4ul of ATP
    • Added 2ul of exonuclease V
  • Tubes were flicked to mix and spun down
  • Tubes were placed in the 37C heat block for 1 hour
  • Then tubes were placed in a 70C heat block for 30 minutes to inactivate the enzyme
  • Samples were kept on ice while awaiting cleanup

I still wanted to see if there was a difference in size retained from the NEB Monarch cleanup or a phenol chloroform cleanup, so I tried both.

NEB Monarch DNA Cleanup

  • Using sample 17 for this
  • Using NEB Monarch PCR and DNA kit protocol
  • Warmed DNA elution buffer to 50C in heatblock
  • Increased the volume in the tube from 40ul to 200ul with 160ul of 10mM tris
  • Added a 2:1 ratio of DNA binding buffer to the tube: 400ul each
  • Inverted tube to mix and spun down
  • Added the total volume (~600) of sample to a spin column
  • Centrifuged the column at 16,000rcf for 1 minute
  • Discarded the flow through
  • Added 200ul DNA wash buffer to the column
  • Centrifuged the column at 16,000rcf for 1 minute
  • Discarded the flow through
  • Added another 200ul DNA wash buffer to the column
  • Centrifuged the column at 16,000rcf for 1 minute
  • Discarded the flow through
  • Centrifuged the column at 16,000rcf for 1 minute “dry”
  • Transferred the column to new labeled 1.5mL tubes
  • Added 15ul of warmed DNA elution buffer to the center of the filter and waited 2 minutes
  • Centrifuged the column at 16,000rcf for 1 minute
  • Placed the sample in the 4C for storage

Phenol-Chloroform Cleanup

  • Using sample 16
  • Basing protocol off of what I’ve done in the hirt extractions (see example and this document)
  • All work was done in the fume hood
  • Increased volume in sample to 300ul with 260ul of 10mM tris
  • 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:
    • 16: 300ul
  • Added 0.1X volume (30ul) of 3M sodium acetate to the tube
  • 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
  • Next day 20240118
  • Centrifuged tube at 4C for 30 minutes at 16,000rcf
    • I did saw a very small pellet
  • Poured 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
  • Poured off the supernatant into the waste
  • Let the tube dry for ~10 minutes upside down
  • Resuspended the pellet in 15ul of 10mM tris
  • Let pellet resuspend for about 10 minutes before use in the gel

20240118 Qubit

  • High sensitivity Qubit of the exonuclease treated samples, there may not really be anything left in these
  • Followed this Qubit protocol
  • 16: 8.78ng/ul
  • 17: 2.22ng/ul

20240118 6 Hour gel

  • Prepared a 0.7% gel with the Seakem agarose that is supposed to be better for gels with HMW DNA
    • 160mL of 1X TAE
    • 1.155g Seakem agarose
  • Microwaved until clear solution
  • Added 3ul of Midori stain and swirled
  • Saved 1mL in a 1.5mL tube and placed in the heat block at 65C to stay warm and liquid
  • Poured the large gel and waited for it to solidify
  • Once solid, added 1 round of PFG marker to the 1st and 8th wells
  • Let the tube of gel cool for 2-3 minutes
  • Filled the wells with the PFG markers with the extra agarose and waited for those to solidify
  • Prepared the samples:
    • 16-exo: 14ul of DNA and 2.8ul of dye
    • 17-exo: 14ul of DNA and 2.8ul of dye
  • Added 48kb ladder and samples into the gel
  • Set the gel for 58V and 6 hours

This looks like the phenol cleanup could yield slightly higher molecular weight DNA, although I am not sure if this indicates intact viral genomes or not, it seems a little small. But I know that after ~50kb they just don’t run very well on the gel. I also found some information online that suggests supercoiled closed circular DNA will run faster in a gel than you’d expect based on size. And this would be the form left from the exonuclease? I think it is best to proceed with using the phenol cleanup method after this treatment in the future.