Team:Hong Kong HKU/Experiments
Notebook
Protocol
General preparation of Native Polyacrylamide gel (Native PAGE gel)
Specifications
A piece of PAGE Gel (~12mL in volume)
Requiring Materials
Storage
Materials (12% PAGE gel)
Quantity
Gel kit
1 Set
Distilled water
6.0 mL
10X TBE
1.2 mL
30% Acrylamide (29:1)
4.8 mL
10% APS
200 μL
TEMED
200 μL
4°C, keep wet in 1x TBE to prevent drying
Steps
1. Clean the glass plates and spacers thoroughly. Rinse the plates withdeionized water and ethanol and set them aside to dry.
2. Assemble the glass plates with spacers in gel caster. Make sure there is no water leakage.
3. Prepare the gel solution according to the desired polyacrylamide percentage. Note the addition of TEMED will immediately trigger the gel to polymerize.
4. Vortex the solution roughly for 5 seconds.
5. Quickly piette the gel solution into the caster. Insert the appropriate comb into the gel, prevent to trap air bubbles under the teeth.
6. Let the polymerization go for 30 minutes at room temperature.
7. Surround the comb and the top of the gel with paper towels that have been soaked in 1x TBE.
Then seal the entire gel in plastic bag and store it at 4°C until needed.
Notes
Different polyacrylamide percentage results in different speed and discrimating ability for sample running down in electrophoresis.
The quantity of the solutions varies with the percentage.
The required quantity of native PAGE gel in common polyacrylamide percentage is listed below.
Polyacrylamide percentage
Distilled water
30% Acrylamide (29:1)
10X TBE
10% APS
TEMED
8%
7.6 mL
3.2 mL
1.2 mL
200 μL
10 μL
10%
6.8 mL
4.0mL
1.2 mL
200 μL
10 μL
12%
6.0 mL
4.8 mL
1.2 mL
200 μL
10 μL
15%
5.2 mL
5.6 mL
1.2 mL
200 μL
10 μL
