# **Gel electrophoresis (Southern)**
Usage amount per slot:
| Material | Concentration | Final concentration | Location |
| :--------- | :-----: | :---: | :----------: |
| PCR product | -- | -- | 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) |
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※ 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**
1. 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
2. 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.
3. Pour the gel into the gel container and wait for solidification (around 15 mins);
## **Step 2: Set up the electrode machine**
1. Prepare the electropheresis machine, the electrode direction should be from negative to positive;
2. After the gel was solidified, put the gel container together with the gel into the elctrophoresis machine;
3. 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**
1. 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;
2. Gently inject 2 μL ladder marker into the first and the last wells of the gel;
3. Gently inject the blank and the PCR products ( around 6 μL ) into the wells;
## **Step 4: Start running**
1. 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**
1. Turn on the machine ( Bio-Rad Gel Doc XR+ ).
2. Put in the gel.
3. Start up the `Image Lab` software.
4. `New protocol` → `Gel imaging`.
6. `Application` → `Select` → `Nucleic acid` → SYBR® Safe.
7. `Position Gel` → `Filter 1` → adjust gel position.
8. `Run protocol`.
9. `Export for publication` ( Export for analysis is 16-bit images ).
Done !!!