Efficient Use of Time in Making Gels

Supplies needed:25 ml graduated cylinder50 μL pipetter
10 ml graduated pipets and bulbs 200 μL pipetter
1 ml graduated pipets125 ml flask

For the bottom gel (enough for two 12% gels):

  1. into 25 ml grad cylinder, pour 28.5 ml of A/B (pour up to brim)
  2. Dump it into the flask. Do not rinse the cylinder
  3. Pour 17.5 ml of buffer into cylinder and dump into flask. Do not rinse the cylinder.
  4. Add 23.4 ml of glass-distilled water to cylinder and dump into flask. Now you may rinse cylinder and put it on the drying rack.
  5. Add 0.7 ml of SDS to the flask
  6. Add 35 μL of TEMED below the surface, and immediately swirl since the TEMED floats to the surface and quickly evaporates if not swirled immediately into solution.
  7. Add 175 μL APS and swirl
  8. Using a 10 ml pipet fitted with a suction bulb, transfer the flask's contents immediately into the forms for making the vertical slabs. Hold a well-comb in position outside the glass and fill the form to about 5 mm below the teeth.
  9. Immediately rinse out the flask and the pipet with tap water.
  10. Add about 0.5 ml of n-butanol to the top of each gel to exclude oxygen. Set some paper towels over the top of the jig so as to keep the fumes of the butanol contained. Butanol is one of the essences of hang-over.
  11. These lower gels solidify in about 15-20 minutes.

For the top gels:

  1. Dump off the butanol in to a sink with much running water. Invert the assembly on a paper towel to drain for a few minutes. (Note: do NOT dispose of the butanol-soaked paper towel in the wastebasket where it will continue to put fumes into the air. Instead, rinse the paper in running water, and then dispose of the paper in the wastebasket. n-Butanol is slightly soluble in water and will go down the drain where the microbes in the sewage treatment plant will metabolize it.)
  2. Using the same 10 ml pipet, add 2.5 ml of top gel Tris to the flask, followed by 6.25 ml of glass-distilled water using the same pipet.
  3. Now add 0.1 ml of SDS; then 6 μL TEMED (refrigerator); then 50 μL APS.
  4. Center combs between the glass panes.
  5. Using the 10 ml pipet again, transfer the contents of the flask into the forms such that the teeth of the combs are about 80% submerged.
  6. Rinse out the pipet and flask
  7. Lift the combs almost out of the forms
  8. Add about 0.3 ml butanol to each form.
  9. Put the A/B and APS away where they belong.
  10. The butanol should have leveled by the time you return. Now carefully push the combs slowly down through the butanol and liquid top gel layers. If you need to center the comb, it much be lifted clear of the liquids first and then pushed down.
  11. Dislodge any butanol bubbles trapped under the teeth by rapidly jerking the frame left and right once or twice.
  12. Cover the works with a plastic bag for the half hour it takes for solidification.
  13. After 30 minutes to overnight, the gel will have formed.
  14. SLOWLY, lift the combs out of the gel. Rinse the combs and set to dry for the next use.
  15. Using a hypodermic needle, tease well-walls vertical if the suction of withdrawing the comb pulled them out of alignment.
  16. Invert the assembly onto a paper towel to drain.
  17. Measure out 400 ml of glass-distilled water, and add to it 100 ml of "5X SDS-PAGE Buffer," mix thoroughly. Pipet some of this to fill the space above the crenelations above the gel up to the top of the panes. (The remainder of the solution will be used later.)
  18. Proceed to the loading of the experimental samples.