12
Table 5.
Distilled Water 4.2 mL
30 % Stock Acrylamide
Solution
0.65 mL
4 X Stacking Gel Tris-
SDS Solution pH 6.6
1.6 mL
10 % Ammonium
Persulphate
67µl
7. Carefully mix the stacking gel solution, avoiding generating air bubbles.
8. Pour off the overlay liquid and rinse the gel with distilled water.
9. Add 6.7µl of TEMED to the stacking gel solution. Mix well. Use a Pasteur pipette
to fill the glass plates up to the top with stacking gel solution.
10. Carefully insert the comb making sure that no air bubbles are trapped under the
ends teeth as these will inhibit sample progression.
11. Allow the stacking gel polymerize for 30 minutes.
For continuous gels:
4. Follow the instructions for mixing the acrylamide solution. Add 15µ of TEMED
and mix well but avoid generating air bubbles.
5. Fill the glass plates to 1 cm below top of notched plate, again avoiding
generating any air bubbles. Filling must be performed quickly before the TEMED
causes the gel to become too viscous.
6. Carefully insert the comb making sure that no air bubbles are trapped under the
teeth as these will inhibit sample progression.
7. Let the gel polymerize. Usually this takes around 15 minutes but this can vary
due to the freshness of the reagents used. If polymerization is taken a lot longer
than this, use fresher stock solutions or add more APS and TEMED.
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