Gel Pouring - VWR shiroGEL Vertical 20 Manual De Instrucciones

Gel Pouring:

For gels with stacking layers:
1. Insert the comb into the glass plates and mark a point on the glass plates 1cm
below where the comb teeth finish. This indicates where to add the resolving gel
to.
2. Add 70µl of TEMED to the gel solution and mix well, but avoid generating air
bubbles.
3. Fill the glass plates again 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 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. Prepare the stacking gel using Table 4 below as a guide. Again stock solutions
are given on pages 22 and 23.
Table 4.
Solution
Distilled Water
30 % Stock Acrylamide Solution
4 X Stacking Gel Tris Solution
10 % Ammonium Persulphate
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. For shiroGEL VERTICAL 20 gels add 26.8µl of TEMED. Mix well. Use a Pasteur
pipette to fill the glass plates up to the top with stacking gel solution.
shiroGEL VERTICAL 20
16.8ml
2.6ml
6.4ml
178 µl
18