Agarose Gel Electrophoresis

1. Buffers and solutions

• Agarose Gel
• Ethidium Bromide
• TAE Buffer
• Gel loading Dye

2. Nucleic acid samples and DNA ladder

3. Equipment and supplies

• An electrophoresis chamber and power supply
• Gel casting trays, sample combs
• Transilluminator

 

Protocol:

1. Prepare appropriate concentration of agarose gel solution in electrophoresis buffer as required: For 25 ml of 2% agarose gel, 0.5mg of agarose was dissolved in 25 ml of TBE (1X) buffer
2. Heat the required amount of agarose gel in TBE until it dissolves. Just keep a watch as agarose solution boils very easily
3. When the molten gel has cooled down, add 0.5μg/ml of ethidium bromide with gentle swirling
4. Pour molten gel into casting tray with appropriate gel combs for forming sample slots
5. Check for the air bubble during pouring of molten gel solution or give a gentle tap to casting tray
6. Allow gel to set completely at room temperature
7. Slightly pour a small amount of electrophoresis buffer on the top of the gel, and remove the combs carefully. Mount the gel in the electrophoresis tank
8. Add enough electrophoresis buffer to cover the gel in the electrophoresis tank
9. To load your samples, take 2 μl of DNA sample and mix it with 0.8 -1 μl of 6X gel loading dye and, slowly load the sample mixture to the slots. Also, load DNA ladder on both the right and left sides of the gel
10. Close the lid of the electrophoresis tank attach the leads to the power supply. Apply a voltage 1-5 V/cm (measured as the distance between positive and negative electrodes)
11. Run the gel until the loading dye/buffer have migrated an appropriate distance through the gel
12. After that, gel tray may be removed and placed directly on a transilluminator to see migrated DNA bands

 

Preparation of solutions:

1. TBE Buffer (10X), pH 8.3

Volume= 500ml

Weigh 54.5 g of Tris base and 15.45g of boric acid in chemical balance and transfer it in the 1000ml beaker. Prepare sodium EDTA solution by weighing 0.925g of Na EDTA (set pH=8.3 using sodium hydroxide pellets) and dissolve it in 40 ml of distilled water. Add EDTA solution to Tris base and boric acid, and adjust 500 ml of volume by adding distilled water.

2. Ethidium bromide 10mg/ml

Add 1g of Ethidium bromide to 100ml of distilled water and stir on a magnetic stirrer for at least 3-4 hours. Either wrap the container of Ethidium bromide or transfer it into a dark bottle.

3. Gel loading dye 6X

Volume= 10ml

For this, weigh 25 mg of bromophenol blue, 25 mg of xylene cyanol FF and 1.5 g Ficoll 400. Transfer them to a screw-capped tube. Add 7ml of deionized/Milli-Q water. Mix until all ingredients are dissolved completely. Further, adjust the volume to 10 ml with deionized/Milli-Q water and mix well.