Polyacrylamide gels

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 water.

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 water.

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.