Prepare Upper (stacking) Gel

Prepare Upper (stacking) Gel
Start procedure 20 min before lower gel is finished polymerizing.

Basic Recipe Table- Upper (stacking) Gel
1 18x16 cm 1.5 mm gel (10 ml)
.
reagent - 3 %
RO H2O (ml) - 4.5
4x Upper Tris Buffer (ml) - 2.5
30/8 % Acrylamide/Bis (ul) - 1.0
10% SDS (ul) -75
APS (ul)


Calculate volumes required and fill in protocol steps below:

1. Add _________ ml RO H2O into a 250 ml side-arm flask using 10 ml pipet with electric pipet filler.

2. Add _________ ml of 4x Upper Tris Buffer using the same pipet
Stored on SDS-PAGE stock solution shelf.

3. Add _________ mL of 30/8% Acrylamide/Bis using a new 10 ml pipet
Stored at 4C
Dispose pipet in HAZARDOUS WASTE container.

4. Add _________ uL of 10% SDS using an automatic pipeter.
Stored on SDS-PAGE stock solution shelf.

5. Add _________ uL of the APS solution using an automatic pipeter.
6. Swirl gently to mix solution without introducing air.
7. Attach flask to vacuum line for 15 minutes to degas.
Secure flask with clamp and use flat surface of oversized rubber stopper to seal flask.

After the Lower Gel has polymerized for 60 min: