Casting Twelve 12% Gels in the Dalttwelve Gel Caster
Casting Twelve 12% Gels in the DALTtwelve Gel Caster
1. Begin by combining the following reagents in 1L sidearm flask:
300 ml 30% Acrylamide
188 ml 1.5 M Tris-HCl pH 8.8
7.5 ml 10% SDS
252 ml ddH2O
2. Place flask on stir plate and add small stir bar. Connect sidearm to vacuum, cover top opening with solid rubber stopper and apply vacuum for 15 min.
3. While solution is degassing, prepare 5 ml of 10% Ammonium persulfate(APS) and assemble DALTtwelve caster. Tilt the caster back so that it rests on its support legs. Place a separator sheet against the back wall to make it easier to remove the last cassette from the unit. Clean each glass plate carefully with ddH2O and Kimwipes. Fill the caster by alternating cassettes with separator sheets. End with a separator sheet then use the thicker spacer sheets to bring the level of the stack even with the edge of the caster. Lubricate the foam gasket with a small amount of GelSeal, place the faceplate onto the caster and screw the black-knobbed screws into place. Do not over tighten the screws.
4. You will need approximately 100 ml Displacement Solution (375 mM Tris pH 8.8, 50% Glycerol, trace of bromophenol blue) and ddH2O to overlay gels. Prepare this while solution is degassing because once the TEMED and 10% APS is added, you must work quickly.
5. After solution is degassed, turn off vacuum, remove stopper and disconnect hose, but keep the flask on the stir plate. Add the following:
3.6 ml 10% APS
0.12 ml TEMED
Continue to stir the solution for 30 seconds then move quickly to the gel caster.
6. Slowly pour the solution into the chamber. The volume prepared is sufficient for 12 gels, so if you are casting fewer, you should make less solution. Pour the solution until it is approximately 3 cm from the desired level. Pour the Displacement Solution into the chamber. Watch the triangular reservoir at the bottom of the caster. When the Displacement Solution has reached the glass plates, place the tube into the grommet to stop the flow. Slowly (drop by drop) overlay each gel with approximately 3 ml ddH2O.
7. Cover the caster with plastic wrap and allow polymerizing over night.
8. After polymerization, carry caster to sink and remove black-knobbed screws. Carefully disassemble and scrape off any access acrylamide into garbage. Rinse outside of cassettes with water to remove any acrylamide particles. Place gels in drying rack.
9. Prepare stacking gel by combining the following reagents:
5.32 ml 30% Acrylamide
10 ml 0.5 M Tris-HCL pH 6.8
0.4 ml 10% SDS
24.4 ml ddH2O
10. Mix thoroughly on stir plate. While mixing, collect 2 transfer pipettes and isobutanol to overlay gels. Add the following reagents only when ready to pour stacker:
0.45 ml 10% APS
0.03 ml TEMED
11. Allow to mix for 30 sec. then, using transfer pipette, place on top of gel.
12. Overlay each gel with 1ml of Isobutanol. Allow to polymerize at least 1 hour.
13. Pour off Isobutanol once polymerized, wash several times with dd water and load gels. Any gels not used need to be kept moist and cool. Wrap gels in damp paper towels, cover with plastic wrap and place at 4 C.