Loading an Agarose Gel, Running it and Analyzing it

Material Required:

Pipettes

Pipette tips

Parafilm

Molecular marker

Loading dye

RNA/ DNA Sample

Ideal loading volumes: 3 to 5μl of sample + 3 to 5μl of loading dye

1 to 2μl of molecular marker + 2 to 3μl of dye

Technique:

1. Place a 1 inch piece of parafilm on the work table.
   
2. Add loading dye.
   
3. To the loading dye on the parafilm, add the DNA sample. Mix well by pipetting.
   
4. Load into a well carefully.
   
5. To load the molecular marker, repeat steps 1 to 4 and replace DNA sample with the molecular marker.
   

Running an Agarose Gel:

Run at 110 to 120 V for 45 minutes to 1 hour. Time will vary. Please look at the position of the leading dye and turn off unit before it runs over.

Imp: While placing the gel, make sure that the wells are on the same side as the black or negatively charged electrodes. Since nucleic acids are negatively charged, they run towards the red or the positively charged electrodes. If the well is placed on the same side as the positively charged electrodes, your sample will run over into the buffer.

Staining an Agarose Gel:

Stain in Ethidium bromide solution for 2 to 4 minutes. Make sure that the stain is on a shaker for efficient staining.

[Since ethidium bromide is a carcinogen, take care while handling it.]

Destain in distilled water or DI water for 15 to 20 minutes.

Nucleic acids will be visible under UV.