Running a 6.5 Hour Gel on the DNA Samples Treated with Exonuclease to Check for HMW DNA

I really need the DiNV DNA to stay intact for the electroporation. While it looks like the exonuclease treatment does increase the ratio of DiNV DNA to cell DNA, I tried two different cleanup methods and I want to know if this has kept the DNA intact. It might be hard to see this, for two of the samples there was basically no DNA, and the other samples have ok amounts, but they might not show up well on the gel. So I added all the DNA I could to this 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: 12ul of DNA and 2.4ul of dye
    • 17-exo: 14ul of DNA and 2.8ul of dye
    • 18-exo: 14ul of DNA and 2.8ul of dye
    • 19-exo: 12ul of DNA and 2.4ul of dye
  • Added 48kb ladder and samples into the gel
  • Set the gel for 55V and 6.5 hours

This gel did not go as well as I’d hoped, there seemed to be just not enough DNA to see very well, and something weird happened to one of the ladders and one of the samples. I edited the photo of the gel to try to see bands clearer, but I’m not sure if that helped.

I see a clear band around 48kb in the sample 18 phenol-cleaned up well, which indicates that some breakage happened because it should be larger than that if intact (although it is not clear that circular DNA moves the same way as linear in a gel so I’m not sure). And I almost see a band that looks larger in the 17 monarch cleanup well, but I can’t really tell.

I really want to be sure if this works and is worthwhile or not, so I will try the treatment, cleanups, and gels again on the cell pellet samples with more input DNA to the exonuclease treatment and see if I can see anything in the gel.