Team:Hong Kong HKU/Experiments
Notebook
Protocol
General preparation of Native Polyacrylamide gel (Native PAGE gel)
Specifications
DNA working solution (1μM)
Requiring Materials
| Materials | Quantity |
|---|---|
| DNA Stock solution (100μM) | 5.0μL/5.5μL |
| 1X TM buffer | 1 Set |
Storage
-20°C
Steps
1. Wipe the bench and gloves with 70% ethanol.
2. Briefly vortex the stock solutions before dilution.
3. Prepare the followings.
| Eppendorf/PCR tube Label | Volume of 1X TM buffer | Volume of 100μM stock solution | Final volume and concentration |
|---|---|---|---|
| O1 (5μM) | 95 μL | 5 μL | 5 μM 100 μL |
| O2 (5μM) | 95 μL | 5 μL | 5 μM 100 μL |
| O3 (5μM) | 95 μL | 5 μL | 5 μM 100 μL |
| O4 (5μM) | 95 μL | 5 μL | 5 μM 100 μL |
| O5 (5μM) | 95 μL | 5 μL | 5 μM 100 μL |
| O1 (25μM) | 16.5 μL | 5.5 μL | 25 μM 22 μL |
| O2 (25μM) | 16.5 μL | 5.5 μL | 25 μM 22 μL |
| O3 (25μM) | 16.5 μL | 5.5 μL | 25 μM 22 μL |
| O4 (25μM) | 16.5 μL | 5.5 μL | 25 μM 22 μL |
| O5 (25μM) | 16.5 μL | 5.5 μL | 25 μM 22 μL |
| Input (5μM) | 95 μL | 5 μL | 5 μM 100 μL |
4. Mix well by tapping followed by briefly centrifuging the eppendorfs.
Notes
TM Buffer is a Tris buffer containing Magnesium ion. Here, we prepare TM Buffer of 40 mM Tris, 100mM MgCl2 and is maintained at pH 8.0 condition.
Refer to the following for the preparation of TM buffer:
| Materials | Quantity |
|---|---|
| MgCl2·2H2O or MgCl2 or MgCl2·6H2O | 0.566g or 0.476g or 1.016g |
| Distilled water | 48 mL |
| 1M Tris solution | 2 mL |
For the steps, it is noted that the pH of the buffer is adjusted to 8 by adding HCl with the aid of a pH meter.
The above quantity is in 2X concentarion, therefore the solution is further diluted to 1X by mixing 115μL 2X TM buffer with 115μL ddH2O to an eppendorf.
Tetrahedron assembly and preparation of DNA solutions for PAGE
General preparation of Native Polyacrylamide gel (Native PAGE gel)
Specifications
A piece of PAGE Gel (~12mL in volume)
Requiring Materials
| 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 |
Storage
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 |
Native PAGE
Steps
Load the followings. (DNA ladder: 2μL, DNA sample: 8μL, 1X loading dye: 8μL)
| Lane | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
|---|---|---|---|---|---|---|---|---|---|---|
| Gel A | 20 bp DNA ladder | O1.6 | O2 | O3 | O4 | O5 | Input | A1 | A2 | A3 |
| Gel B | 20 bp DNA ladder | A4 | A5 | A6 | A7 | A8 | A9 | A10 | B1 | B2 |
| Gel C | 20 bp DNA ladder | B3 | B4 | B5 | B6 | B7 | B8 | B9 | B10 | 1X Loading buffer |
| Gel D | 20 bp DNA ladder | C1 | C2 | C3 | C4 | C5 | Tetra | Tetra+Input | Output | 1X Loading buffer |
Run the gel at constant voltage at 100V for until the bands of dye reach ¾ of the length of the gel.
Preparation of agarose gel
Specifications
A piece of 1% Agarose gel
Requiring Materials
| Material | Quantity |
|---|---|
| Agarose powder | 1 g |
| 1X TBE | 100mL |
Storage
4°C
Steps
1. Pour 1g agarose powder into microwavable flask along with 100mL of 1xTBE.
2. Put into microwave for 1-3 minute until the agarose is completely dissolved. A nice rolling boil will be observed upon competion.
3. Let agarose solution cool down in room temperature for 5 minutes.
4. Pour the agarose into a gel tray with the well comb in place.
5. Place newly poured gel at 4°C for 10-15 minutes OR let sit at room temperature for 20-30 minutes, until it has completely solidified.
Agarose gel electrophoresis
Specifications
A piece of 1% Agarose gel
Requiring Materials
| Material | Quantity |
|---|---|
Steps
1. Cast the gel into position.
2. Fill the gel box with 1X TBE until the gel is covered by the buffer.
3. Load the DNA loading dye to samples.
4. Load the followings.
| Lane 1 | Lane 2 | Lane 3 | Lane 4 | Lane 5 | Lane 6 | Lane 7 | Lane 8 | Lane 9 | Lane 10 | Lane 11 |
|---|---|---|---|---|---|---|---|---|---|---|
| 100bp DNA ladder | DNA ladder (2 log) | O1 (1μM) | O2 (1μM) | O3 (1μM) | O4 (1μM) | O5 (1μM) | Input (1μM) | O5+Input (1μM) | Tetra (1μM) | Tetra+Input |
