12
3.
Fill the glass plates smoothly till the mark avoiding generating any air
bubbles. Filling must be performed quickly before the TEMED causes
the gel to become too viscous.
4.
Overlay the gel extremely carefully with 1 ml of Isobutanol, Isopropanol
or distilled water. When using distilled water extra care must be taken
to ensure there is no mixing with the gel solution.
5.
Let the resolving gel polymerize. Usually this takes around 15 to 30
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.
6.
Let the resolving gel polymerize. Usually this takes around 15 to 30
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.
7.
Prepare the stacking gel solution.
8.
Before casting the stacking gel, insert a piece of filter paper to dry the
area in between the glass plates above the resolving gel. Take care
not to touch the surface of the gel.
9.
Carefully pour the stacking gel solution, avoiding generating air
bubbles.
10.
Carefully insert the comb making sure that no air bubbles get trapped
under the ends of the comb teeth as these will inhibit sample
progression.
11.
Allow the stacking gel to polymerize.
12.
Once the gel is polymerized it is ready for the electrophoresis run.
Casting a gel without stacking layer
•
Prepare the resolving gel solution. Mix well and avoid generating air
bubbles.
•
Pour the solution smoothly into the glass plates avoiding any air bubbles
until the top of the notched glass plate is reached.
•
Carefully insert the comb making sure that no air bubbles get trapped
under the ends of the comb teeth as these will inhibit sample
progression.
