Gel Electrophoresis Experiment

Theory Agarose gel electrophoresis is a widely used procedure in various areas of biotechnology. This simple and precise analytical procedure is used in the research, biomedical and forensic laboratories.

It is used for (i) determining the size of DNA molecules in the range of 500 to 30,000 base pairs, (ii) to analyze DNA fragments generated by restriction enzymes, and (iii) to separate other charged biomolecules such as dyes, RNA and proteins.

Principle

DNA molecules are negatively charged at neutral or alkaline pH and migrate towards anode when an electric field is applied. Charge/ mass ratio of nucleic acid is unity, thus migration occurs largely on the basis of molecular size of the DNA molecules.

Material

Minigel horizontal agarose

Gloves must be woven while preparing this solution

Plasmid DNA preparation. To 20 μl of plasmid DNA preparation add 10 μl of gel loading solution and mix properly

Standrad DNA marker: Take 20 μl of the  Hind III DNA digest, add 10 μl of gel loading solution and mix them well.

Method

1. Take a clean dry gel casting plate and make a gel mould using an adhesive tape along the sides of the plate to prevent running off of the material to be poured on the plate

2. Pour 50 ml of 1% agarose solution kept at 50ºC onto the casting plate. Immediately place the supplied comb about 1 cm from one end of the plate ensuring that teeth of the comb do not touch the glass plate. Wait till a firm layer of gel is

Carefully remove the comb and tape surrounding the plate and transfer the gel plate to the electrophoretic tank such that wells are towards the cathode

4. Pour 1X TAE buffer into the tank until the gel is completely submerged. Connect the electrode to the power supply

5. Load the plasmid DNA preparation and standard DNA marker into separate well with help of a micropipette or a syringe

6. Turn ‘ON’ the power supply and run at 100 V (10-15 mA). Monitor the progress of movement of tracking dye (bromophenol blue) during electrophoresis.

7. Turn ‘OFF’ the power supply when the tracking dye has reached near the opposite edge of the gel.

8. Transfer the gel from casting plate onto a UV-transparent thick plastic sheet and place it in a staining tray containing ethidium bromide solution for 20-30 min.

9. For destaining the gel, place it in water for 15-20 min.

10. Now place the gel along with UV-transparent sheet on a UV-transillumintor and view the gel in UV light for presence of orange colored bands.

11. Measured the distance moved by each band from edge of the loading well. Draw a graph of log10 Mw of standard DNA marker vs the distance travelled by each of them

12. From the distance travelled by supplied DNA preparation, determine its molecular weight using the calibration