Calculate the amount of agarose needed based on the desired gel concentration and size. Typically, 1% agarose is used for most DNA separations.
Weigh the appropriate amount of agarose powder and add it to a flask containing electrophoresis buffer.
Heat the flask in a microwave or a water bath until the agarose is completely dissolved.
Allow the agarose solution to cool down until it is still liquid but can be touched comfortably.
2.Prepare the gel mold
Place the gel mold (e.g., a gel tray or a casting tray) on a level surface.
Clean the gel mold and the surrounding area to prevent contamination.
Set up the comb in the desired location within the gel mold.
3.Pour the gel
Mix the agarose solution gently to ensure it is homogeneous.
Add ethidium bromide to the agarose solution if you want to visualize DNA bands using UV light (follow the manufacturer's instructions).
Carefully pour the agarose solution into the gel mold, ensuring it covers the comb and there are no air bubbles trapped.
Allow the gel to solidify completely for about 20-30 minutes.
4.Prepare the samples
Mix the DNA samples with gel loading dye in a 1:1 ratio (e.g., 5 μL of sample + 5 μL of loading dye).
Mix the DNA ladder with gel loading dye according to the manufacturer's instructions.
5.Load the gel
Remove the comb gently from the solidified gel, creating wells for sample loading.
Carefully load the DNA ladder into one well.
Load the DNA samples into separate wells.
If needed, include a well containing only the gel loading dye as a negative control.
6.Run the gel
Carefully place the gel into the electrophoresis apparatus, ensuring that the wells are closer to the negative electrode (black) and the DNA will migrate towards the positive electrode (red).
Fill the electrophoresis tank with enough electrophoresis buffer to cover the gel.
Connect the leads of the power supply to the electrodes of the electrophoresis apparatus.
Set the desired voltage (typically 100-150 V) and run the gel for the recommended time based on the expected DNA size and gel concentration (usually 30-60 minutes).
7.Visualize the DNA
Once the run is complete, carefully remove the gel from the apparatus.
If you stained the gel with ethidium bromide, visualize the DNA bands under a UV transilluminator.
Alternatively, you can use other DNA stains or gel documentation systems for visualization.
Remember to follow appropriate safety precautions while working with DNA stains, UV light, and electrical equipment. Always refer to the specific manufacturer's instructions for the equipment and reagents used in your experiment, as protocols might vary slightly.
