Gel electrophoresis (Southern)
Preparation
| Material |
Concentration |
Final concentration |
Location |
| PCR product |
-- |
-- |
Store at 4°C |
| TAE buffer ※ |
50X |
0.5X |
II |
| Agarose gel † |
-- |
3% |
II |
| SYBR® stock solution * |
1000X |
1X |
II |
| Loading Dye |
6X |
1X |
4°C (c) |
| RTU-100 (Ladder marker) |
-- |
-- |
4°C (c) |
|
|
|
|
※ To prepare the 0.5X TAE buffer, make at least 1 L, as each run requires at least 500 mL 0.5X TAE buffer;
† The big gel slot requires around 40 mL gel solution; the small gel slot requires 20 mL gel solution. For example, to make a 3% big gel, add 1.2 g agarose powder (ultra pure grade) to 40 mL 0.5X TAE buffer.
* To prepare 1000X SYBR stock solution, dilutes 100 µL SYBR® raw solution with 900 µL DMSO. This chemical is used to stain the DNA and detected by UV radiation.
Procedure
Step 1: Make gel
- Prepare the gel. After adding agarose powder into the 0.5X TAE buffer, melt them with microwave. Gently shake the vial during each time points.
- use mid-low firepower, 1 mins → 30 secs → add ddH2O → 20 secs → 10 secs
- Add 0.001 volume of SYBR® stock solution into the gel solution immediately ( 1000X → 1X ), gently shake to mix them well;
- For example, 40 mL gel requires 40 µL 1000X SYBR stock solution.
- Pour the gel into the gel container and wait for solidification (around 15 mins);
Step 2: Set up the electrode machine
- Prepare the electropheresis machine, the electrode direction should be from negative to positive;
- After the gel was solidified, put the gel container together with the gel into the elctrophoresis machine;
- Add some TAE buffer (same strength with the gel; 0.5X in this case) into the electropheresis machine
and make sure the gel was fully submerged in the buffer;
Step 3: Load sample
- Add loading dye to the PCR product. The loading dye final concentration should be 1X.
For example, 2 µL 6X loading dye + 10 µL PCR product;
- Gently inject 2 µL ladder marker into the first and the last wells of the gel;
- Gently inject the blank and the PCR products ( around 6 µL ) into the wells;
Step 4: Start running
- Set the voltage as 135 V and the runtime as 40 mins;
5 ~ 10 V / cm for DNA size < 1 kb;
4 ~ 10 V / cm for DNA size between 1 ~ 12 kb;
1 ~ 3 V / cm for DNA size greater than 12 kb.
For our machine, the distance between the electrodes is around 13.5 cm.
Step 5: Gel-imaging
- Turn on the machine (Bio-Rad Gel Doc XR+);
- Put in the gel;
- Start up the "Image Lab" software;
- New protocol;
- Gel imaging;
- Application → Select → Nucleic acid → SYBR® Safe;
- Position Gel → Filter 1 → adjust gel position;
- Run protocol;
- Export for publication ( Export for analysis is 16-bit images ).