Question

A mixture of fragmented DNA was electrophoresed in an agarose gel. After staining the gel with ethidium bromide, no DNA bands were observed. What could be the reason?

Answer 1

Agarose gel electrophoresis:

Agarose gel electrophoresis is the process of separation of DNA fragments in an electric field based on their size.

$\bullet$ The DNA fragments and size markers/ ladder are loaded onto the gel well and current is passed through the gel.

$\bullet$ The DNA being negatively charged, moves towards the positive electrode during gel electrophoresis.

$\bullet$ The smaller the size of the molecule, the faster and farther it travels. This is how the fragments are separated.

Visualisation of DNA in agarose gel:

To visualise DNA in the agarose gel, a positively charged dye ethidium bromide (EtBr) is added in the agarose gel or electrophoresis buffer.

$\bullet$ This dye intercalates itself into the DNA backbone and gives orange fluorescence upon the exposure to the UV light.

$\bullet$ Thus, DNA is visualised by the exposure of UV light and orange bands of DNA are observed on the agarose gel.

The reason for not observing DNA bands:

The reasons behind not observing DNA bands after agarose gel electrophoresis could be:

$\bullet$ Problems in sample loading
$\bullet$ Wrong orientation of electrodes
$\bullet$ Improper staining

Problem in sample loading:

DNA samples that were loaded on the gel may have been contaminated with nuclease enzymes which might have degraded the separated molecules. Alternatively, the concentration of DNA loaded into the wells could be too small which was not enough to form visible bands.

Orientation of electrodes:

Electrodes might have been put in opposite orientation in the gel assembly that is anode towards the wells (where DNA sample is loaded). Since DNA molecules are negatively charged, they move towards anode and hence might have moved out of the gel instead of moving into the matrix of gel.

Concentration of EtBr:

Ethidium bromide might not have been added in sufficient quantities for the DNA to be stained appropriately.