11
Table 3: Preparation of the resolving gel solution for two 10 x 10cm gels using
1 mm spacers.
Solution
5%
7.50%
10%
12%
15%
17.5%
Distilled
Water
8.7
mL 7.
5mL
6.3
mL 5.25
mL 3.75mL 2.5
mL
30 % Stock
Acrylamide
Solution
2.5 mL
3.75 mL 5 mL
6 mL
7.5 mL
8.75mL
4 X Tris-SDS
Solution pH 8.8
3.75
mL 3.75
mL 3.75
mL 3.75
mL 3.75
mL 3.75mL
10 %
Ammonium
Persulphate
150 µl 150 µl 150 µl 150 µl
150 µl
150 µl
Gel Pouring:
For discontinuous gels:-
1. Insert the comb between the glass plates and mark a point on the glass plates
1cm below the bottom of the teeth. This is the level for the resolving gel.
2. Add 15 µl of TEMED to the resolving gel solution.
3. Fill the glass plates to the line and avoid generating any air bubbles. Filling must
be performed quickly before the TEMED causes the gel to become too viscous.
4. Carefully, overlay the gel with 1 ml of 1% 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. Allow the resolving gel to polymerize. This usually takes 15 to 30 minutes, but
can vary due to freshness of reagents used. If polymerization time is excessive,
use a fresh stock solution or add more APS and TEMED.
6. Prepare the stacking gel using Table 5 below as a guide. (See appendix for
stock solutions.)
