ACRYLAMIDE GEL PROCEDURE
Gel Cassette Assembly (Bio-Rad Mini Protean 3)
-
Clean and completely dry the glass plates, combs, and any other pertinent
materials.
-
Place a short plate on top of a spacer plate. Insert both plates
into the green casting frame on a flat surface. Be sure that the "legs"
of the casting frame are down. Clamp the casting frame and check that the
plates are level on the bottom.
-
Place the cassette assembly on a flat surface so the plates are level.
-
Determine the appropriate gel type and composition for your experiment.
Combine all reagents (except TEMED) in small beaker in the order listed.
Table 1. Denaturing acrylamide gel conditions
| Gel Composition |
6%
|
8%
|
10%
|
12%
|
| Water (mL) |
6.4
|
5.9
|
5.4
|
4.9
|
| 5X TBE (mL) |
2.0
|
2.0
|
2.0
|
2.0
|
| Ureaa (g) |
4.8
|
4.8
|
4.8
|
4.8
|
| 40% acrylamide (mL) |
1.5
|
2.0
|
2.5
|
3.0
|
| 10% APS (mcL) |
100
|
100
|
100
|
100
|
| TEMED (mcL) |
10
|
10
|
10
|
10
|
| Total Volume (mL) |
10
|
10
|
10
|
10
|
a the best practice would be
to dissolve the urea in a
small volume of water, add the buffer and acrylamide
then adjust the volume to 10 mL
Table 2. Native acrylamide gel conditions
| Gel Composition |
6%
|
8%
|
10%
|
12%
|
| Water (mL) |
6.4
|
5.9
|
5.4
|
4.9
|
| 5X TBE (mL) |
2.0
|
2.0
|
2.0
|
2.0
|
| 40% acrylamide (mL) |
1.5
|
2.0
|
2.5
|
3.0
|
| 10% APS (mcL) |
100
|
100
|
100
|
100
|
| TEMED (mcL) |
10
|
10
|
10
|
10
|
| Total Volume (mL) |
10
|
10
|
10
|
10
|
Table 3. SDS-PAGE gel conditions
| Gel Composition |
6%
|
8%
|
10%
|
12%
|
| Water (mL) |
6.4
|
5.9
|
5.4
|
4.9
|
| 1.5M Tris (mL) |
2.6
|
2.6
|
2.6
|
2.6
|
| 10% SDS (mcL) |
100
|
100
|
100
|
100
|
| 40% acrylamide (mL) |
1.5
|
2.0
|
2.5
|
3.0
|
| 10% APS (mcL) |
100
|
100
|
100
|
100
|
| TEMED (mcL) |
10
|
10
|
10
|
10
|
| Total Volume (mL) |
10
|
10
|
10
|
10
|
-
Add TEMED only when completely ready for polymerization to occur!
-
When ready to pour the gel, quickly add the TEMED, mix by swirling
gently, draw the solution into a 10 mL syringe and gently dispense the
solution between the glass plates completely filling the space between
the plates. Insert the well forming comb into the opening between the glass
plates.
-
Eject the remaining acrylamide solution back into the small beaker.
Polymerization of this solution indicates the completeness of polymerization
of the gel between the plates.
-
Allow gel to polymerize (~30 min)
-
Once the gel has polymerized, the comb can be gently removed. The polymerized
gel between the short plate and spacer plate forms the "gel cassette".
Electrophoresis
-
Remove the gel cassette from the casting stand, remove comb and place it
in the electrode assembly with the short plate on the inside. Place buffer
dam plate opposite the gel cassette assembly.
-
Provide a slight upward pressure on the gel cassette and buffer dam while
clamping the frame to secure the electrode assembly. This step is important
to minimize potential leakage during the electrophoresis experiment.
-
Place the assembly into the electrophoresis tank
-
Prepare 500 mL 1X electrophoresis buffer
-
Completely fill the inner chamber with 1X electrophoresis buffer.
Check for leaks.
-
Use a gel-loading tip or syringe to pipette buffer into each well to remove
debris.
-
When all wells are sufficiently cleaned, using a gel loading pipette tip,
slowly pipette a maximum of 10 mcL of sample or molecular weight marker
into individual wells. A yellow guide can be placed on top of the electrode
assembly to aid in loading the gel.
-
Add enough 1X electrophoresis buffer to the region outside of the frame
to cover the bottom of the gel cassette assembly.
-
Cover the tank with the lid aligning the electrodes (black or red) appropriately.
-
Connect the electrophoresis tank to the power supply.
-
Run the gel at the appropriate voltage, power or current for your particular
application.
-
When electrophoresis is complete, turn off the power supply, disassemble
the apparatus, and clean all relevant equipment.
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