Gel loading and electrophoresis

  1. Make 2.3 litres of 0.5X TBE buffer (230 ml 5X TBE + 2070 ml H2O).
  2. Add 1.0 g Seakem Gold agarose to 100 ml 0.5X TBE Buffer.
  3. Carefully melt agarose in microwave oven. Save 5 ml (at 55oC) to seal the wells.
  4. Assemble the gel forming tray, assure that the tray is level with the well comb in place, and pour melted agarose into forming tray (cool melted agarose to 55-60oC before pouring).
  5. Allow gel to solidify for a minimum of 30 min.
  6. Add remaining 2.2 litres of 0.5X TBE buffer to the CHEF electrophoresis box.
  7. Chill the buffer to 14oC by circulation through the chiller-pump unit.
  8. Adjust flow rate of buffer through the chiller-pump unit to between 0.75 and 1.0 l/min.
  9. Once gel has solidified and the comb is removed, remove a digested plug slice from the microcentrifuge tube.
  10. Place the slice onto inner surface of sterile petri dish.
  11. Carefully slide the plug slice onto the side of a sterile razor blade using a sterile microspatula.
  12. Holding the razor blade (with the plug slice sticking to the side) at the edge of a well, carefully use the microspatula to slide the plug slice into the well. Use care not to introduce air bubbles in the well.
  13. Wash and flame sterilize the razor blade and microspatula for continued loading of wells with other prepared DNA plug slices.
  14. Add low range molecular size standards (4.9 – 120 kb)(Bio-Rad) to outer wells.
  15. Seal the wells with the saved SeaKem Gold agarose. Use care not to introduce any air bubbles during sealing and fill any wells that do not contain slices.
  16. Place the gel into the circulating pre-cooled buffer within the CHEF electrophoresis box.
  17. Allow the gel to cool for 15 min prior to beginning electrophoresis.
  18. Perform electrophoresis in the Bio-Rad CHEF DR III system using the following electrophoresis run parameters:
  • Switch angle: 120o
  • Switch time: 1 - 6 sec
  • Voltage:  6 V/cm
  • Temperature: 14oC
  •  Run time: 16 hours

  19.  Following electrophoresis, remove and stain the gel in the dark with Sybr Green (Molecular Probes, Inc., Eugene, OR) (30 µl of 10,000X concentrate diluted into 300 ml TE pH 7.5) at RT for 45 min.

  20.  Photodocument the gel via UV transillumination/epi-illumination (254 nm).