Edited by Chang Zhu
1. Wash the gel plates, spacers, and combs with soap. Rinse with water.
2. Rinse or wipe the plates with 70% ethanol and let air-dry.
3. Assemble the gel apparatus, sandwich the spacers between a
pair of gel plates (one shorter than the other).
4. Fit the gel sandwich in the gel caster or clamp the plates on
three side except the top with binder clips.
For small plates, spacers at the bottom and sides may be sufficient
to prevent any leak. Vasline may be used on spacers to prevent leak.
For big gel plates, it may be necessary to seal the bottom and sides
with an electrical tape or molten 2% agarose.
5. (optional) Test if the assembled apparatus has any leaks using
6. Make separating gel. For 8 cm x 10 cm mini-gel, make 10 ml separating
gel solution per gel. 15 ml will be sufficient for two min-gels. Add TEMED just before pouring the gel.
Calculate each component needed using
PAGE/SDS --- recipe calculator or BioToolKit SDS-Page calculator.
7. (optional) De-gas the separating gel solution. This is usually not
ncessary for small gels.
8. Pour the separating gel. Leave enough space (~1/4) on the top for
stacking gel. Pour the gel along one side with a 5 ml pipette. Pour
slowly and continuously to avoid trapping any air bubbles.
9. Overlay the separating gel with water or water saturated 1-butanol.
10. Allow the gel to polymerize ( ~ 30 minutes). Ammonium persulfate
solution needs to be fresh (less than a couple of weeks old). Polymerization
is faster when fresh AP is used.
11. After the separating gel has polymerized, rinse the gel top with
12. Make 5 ml stacking gel solution.
13. Pour the stacking gel all the way to the top.
14. Carefully insert comb and avoid trapping any air bubbles.
15. Allow the gel to polymerize.
16. Remove the bottom spacer or tape.
Assemble on the running apparatus, the shorter side of the gel
facing running buffer tank.
17. Fill the tanks with 1X SDS-PAGE running buffer, remove
and clean the wells with a needle connected to a syringe if necessary.
18. Load denatured (boiled) samples with loading buffer, and run
at a constant current of 25-30 mA until the bromophenol blue front has reached
the end. Double the current if two gels are running.