MODIFIED ECKHARDT GEL PROCEDURE (L. Unger)

MODIFIED ECKHARDT GEL PROCEDURE (L. Unger)

  1. Grow strains in YEP broth to a Klett reading (#66 filter) between 30 and 70.
  2. Harvest volume of cells that will correspond to a Klett value of 42. 42/Klett reading = ml of cells to harvest.
  3. Pellet cells in microfuge, High speed 1-4 min at room temperature.
  4. Decant supernatant, tap out as much liquid as possible without dislodging pellet.
  5. Add 1 ml 1 M NaCl, resuspend pellet with microglass rod, vortex well. (vortex with 10 short counts of 1, and one long vortex to a count of twenty)
  6. Pellet cells 4 min high speed at room temperature, decant supernatant as before. Pellets can be easily dislodged at this stage, so take caution. Try to remove as much supernatant as possible with Kimwipe.
  7. Place samples on ice while preparing gel apparatus.
  8. Re-melt prepared 0.7% agarose. Place small amount in assembled gel unit and move melted agarose around to seal all edges. Let agarose cool a bit and add rest to unit without forming air-bubbles. Clamps around glass should have pressure at the edge of the plates. After gel solidifies, clamps may be pushed in. Insert comb and let melted agarose over-run. Let solidify.
  9. Add 120 l 20% Ficoll in TB with 1 g/ml RNase A to pellets.
  10. Flush wells of vertical electrophoresis apparatus with TB to dislodge agarose fragments left after removing comb. Be careful not to disturb bottom of slot and remove as much liquid as possible before adding samples.
  11. To samples add 20 l TE, resuspend with microglass rod, vortex (as in 5).
  12. To each sample separately, add 20 l pronase (10 mg/ml), mix gently and transfer quickly with micropipette to gel well. Let sample sit in well for 30 min before next step. Follow time by stopwatch.
  13. Add 120 l 10% Ficoll, 1% SDS, 0.005% BPB in TB to sample in well by layering. Partially mix with 6 gentle strokes of a toothpick in the well.
  14. Add 120 l 5% Ficoll, 1% SDS in TB to form a third layer.
  15. Seal wells by filling with melted agarose (slightly cooled) Use Pasteur pipette.
  16. Fill electrophoresis unit with TB. Initially run gel for 75 min at 7mA.
  17. Run gel for 4.5 hrs at 40 mA (100V)
  18. Stain gel in 0.4 g/ml EtBr in TB for 10 min.
  19. Destain gel in TB for 20 min.
  20. Photograph in uv light.

Materials:

Yeast Extract Peptone Broth (YEP): 1% peptone, 1% yeast extract, 0.5% NaCl; pH to 7.1 with NaOH; autoclave.
TE buffer: 50 mM Tris (pH 8), 20 mM EDTA
10X Tris borate buffer (pH 8.2): Tris base 108 g; EDTA 9.3 g; Boric acid 55 g; ddH20 to 1 L; filter sterilize.
2M Tris buffer (pH 8)
0.5 M EDTA (pH 8)
5 M and 1 M NaCl
3 M Sodium acetate (pH 5.2)
20% SDS
Ethidium Bromide, 10 mg/ml
RNase 2 mg/ml (prepared in 1 ml aliquots): RNase 10 mg in 5 ml 0.01 M NaAc, 0.15 M NaCl; heat to 80 C for 10 min.
Pronase, 10 mg/ml: 50 mg Pronase in 5 ml TE buffer (pH 8); heat at 37 C for 60 min.
0.1% bromphenol blue (BPB)
20% Ficoll 400 in TB buffer
22% Ficoll 400 in TB buffer
10% Ficoll 400, 1% SDS, 0.005% BPB, in TB buffer
5% Ficoll 400, 1% SDS, in TB buffer
0.7% agarose

capillary pipettes calibrated to 40 l and 120 l
toothpicks (flat)

Vertical electrophoresis unit: DAN KAR Corp., PO Box 279, Reading, Mass. 01867, (617)944-1550. Model E?
gel dimensions: comb - 4 mm thick, 10 wells 9 mm wide each, 15.5 mm high.
glass plate (notched) - 11.8 cm long, 16.5 cm wide
glass plate (regular) - 14 cm long, 16.5 cm wide.

optional: Gilson-Microman positive displacement pipette 50-250 ul
ca. $123 for unit, $35 for replacement tips (pistons).