Testing Running a Gel for HMW DNA using the NEB MidRangePGF Marker, Trying to Find a Way to Separate Out the Ladder, 16 hours at 4 degrees and 140V

Notes

  • Rob found this paper where they use a FIGE system (field inversion gel electrophoresis) at 4 degrees C, 135 volts, and I think 16 hours (the specifications for how they run it are specific to the FIGE machine, but it says 8 hrs twice, so I thought that meant 16 hrs), also this paper about FIGE uses a 12 hr run time
  • We don’t have a FIGE machine, they switch the direction of the current every few seconds it looks like, and in a specific time ratio or current ratio: “Two basic electrophoretic modes can be used in order to achieve a net migration in this configuration: The same voltage (V) can be applied for a longer time (T) in the forward (TF) than in the reverse direction (TR). In general, the ratio TF/TR is kept constant over the course of the experiment to achieve maximum resolution; or: A higher voltage can be applied in the forward (VF) than in the reverse (VR) direction but for the same amount of time (TF= TR)” - x
  • I used 140V because [this paper(https://link.springer.com/protocol/10.1385%2F0-89603-229-9%3A3) uses 9.5V/cm, we have 15cm long gel, so that’s 145V, and the other paper used 135V, so 140 was inbetween

Making and Loading the gel, 20211109

  • 1% gel mix:
    • 180mL 1X TAE
    • 1.8g agarose
  • Microwaved for ~4min
  • Pipetted 1mL into a 1.5mL tube and saved on the heat block at 65 degrees C make sure the heat block is on and running
  • Made sure the large tray had well sealed tape barriers
  • Let the gel liquid cool for ~10 min until pouring into the tray and placing in the combs
  • Gel cooled in ~20 min or less
  • I placed the gel box in the fridge at this time to cool it down
  • Added 1 round of PFG ladder to the right most well
  • Let the 1mL saved gel cool for ~3 min outside of the heat block
  • Filled the PFG well with cooled gel liquid to the top of the well to seal the rounds in
  • Waited ~3 minutes for the wells to cool
  • Made up 48kb ladder:
    • 1.2ul ladder
    • 6ul loading dye
    • 28.8ul molecular grade water
  • Took the gel box out of the fridge and placed the gel tray in the gel box (without tape)
  • Added 16ul 48kb ladder to the well right next to the PGF well
  • Added 6ul 1kb plus ladder to the well right next to the 48kb well (3rd well)
  • Placed the gel box back into the fridge, at the bottom level. I adjusted the screw things to try to make it as level as possible
  • I put the gel power station in the fridge as well
  • Set the timer for 16 hours at 140 volts and started it at ~5pm

Imagine the Gel 20211110

  • Stopped the gel the next morning at 9:26am
  • Sliced the gel for easy handling, and placed in the EtBr bath for 1 hr
  • After being in the EtBr, placed the gel on the UV for imaging
  • HA! Nothing left on the gel!

After going back and looking at the 22hr gel that was at 40V(see image below), even the largest DNA is halfway down the gel. Even though the time was less and the gel % more, I think the increased voltage by ~100V made everything move soooo much faster.

Another interesting thing was that I noticed when I took the gel box out of the fridge that it was hot. So I measured the temp and it was 35 degrees C inside the gel box. So it wasn’t actually that cold in there, but probably colder than it was without being in the fridge.