The underlying protocol is followed for a typical agarose gel electrophoresis for the resolution and recovery of large DNA fragments using horizontal electrophoresis tanks. 

Please ensure that the horizontal electrophoresis tank, gel tray, sample comb, gel maker and other components are clean and dry before gel preparation.

1. Preparation

Weigh an appropriate amount of agarose into an Erlenmeyer flask, add an appropriate amount of buffer, place it in a water bath, a magnetic heating stirrer or a microwave oven and heat until it is completely melted, shake well and make an agarose gel solution.

2.Gel Making

Place the gel maker horizontally on the experimental bench, select a suitable gel tray (different sizes of gel trays are selected according to different experimental needs, the specifications of the gel tray are 120mmX120mm , 120mmX60mm , 60mmX120mm , 60mmX60mm), put the gel tray Place it in thegel maker (two pieces of 60mmX60mm gel can be made at the same time, and only one piece can be made for other specifications) and put the sample comb in a fixed position. Add 5ul SerRed (or other nucleic acid dyes) solution to the agarose gel cooled to about 55°C . After mixing, carefully pour it into the gel tray, and slowly spread the gel until the entire surface of the gel tray is uniform. For thegel layer, let stand at room temperature until the gel is completely solidified, and then gently pull out the sample comb vertically to complete the preparation of thegel plate.

3.Sample Loading

Take the prepared gel and the gel tray out of the gel maker together and place them in the electrophoresis box with the sample addition hole close to the negative electrode (black is the negative electrode). Add running buffer until the gel plate is covered.

Use a 10ul micropipette to add the samples to the sample grooves of the rubber plate respectively. After each sample is added, a sample addition head should be replaced to prevent contamination. When adding samples, do not damage the gel surface around the sample wells (pay attention to the order of adding samples) .

4.  Electrophoresis

Cover the top cover according to the correct position of the positive and negative poles (red is positive, black is negative), insert the electrophoresis wire into the electrophoresis power supply according to the correct color, and select the appropriate voltage to start electrophoresis (the specific electrophoresis parameters are adjusted according to the actual experimental parameters) .

The sample moves from the negative (black) to the positive (red) direction. As the voltage increases, the effective separation range of the agarose gel decreases. The electrophoresis was stopped when the bromophenol blue moved to about 1 cm from the lower edge of the gel plate.

5.  Observation

Thisintegrated horizontal electrophoresis instrument is equipped with a blue lightlight transilluminator, which can observe the electrophoresis situation in real time with ( SPW-6S electrophoresis power supply ).

Gel Preparation

1. Prepare a sufficient volume of electrophoresis buffer to prepare gel and fill the horizontal electrophoresis tank. The buffer used is usually TAE or TBE. Add 0.5µg/ml of ethidium bromide to the electrophoresis buffer to facilitate DNA visualization.
2. Mix an adequate amount of electrophoresis grade agarose with the buffer to construct the gel. Melt agarose and swirl to ensure uniform mixing. 
3. Seal the gel casting tray using tape, pour in the melted agarose, and insert the combs. Ensure no bubbles are trapped under the combs, and all bubbles on the gel surface are removed before the gel sets.
4. Remove the tapes once the gel hardens and withdraw the combs without tearing the wells. Most gel platforms allow 0.5-1mm of agarose to remain between the comb’s bottom and the base of the gel platform. This ensures that sample wells are completely sealed and prevents the tearing of wells while removing the combs. 
5. Place the gel casting tray containing set gel in the electrophoresis tank and add a sufficient volume of electrophoretic buffer to cover the gel up to 1mm deep. Avoid air pocket formation within the wells. 
6. Prepare the DNA samples in a volume that does not overflow the wells after adding 10x loading buffer. Use a micropipette to load the samples into the well. Avoid mixing samples. Include appropriate DNA molecular weight markers.
7. Attach the leads and set the voltage to the desired level (normally 1-10V/cm of gel) to run the gel. The dyes present in the loading buffer help monitor the migration of DNA fragments. 
8. Switch off the electricity supply when bromophenol blue from the loading buffer has moved a distance sufficient for the DNA separation. If EtBr is incorporated into the gel, you can directly visualize DNA under a UV source (Voytas, 2000).