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)

^※ 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 ).