Question

Why does the 1.6 kb ladder standard band appear twice as bright as it should? In answering this question, consider that the standards decrease in size from top to bottom and Gel Red stained brightness decreases proportionately.

Answer 1

Ethidium bromide (EtBr ) is used in the agarose gel to visualize DNA molecules. It is a fluorescent dye. The EtBr molecules intercalate between the DNA bases and exhibit fluorescence under UV exposure. EtBr shows 50 times better binding capacity towards double stranded DNA as compared to single stranded DNA. Nucleic acids are degraded by longer UV exposure therefore; longer exposure may lead to less sharp bands.

High-intensity bands in the DNA ladder (eg. 1.6 kb): These bands are the reference bands. Their intensity is supposed to be high. Those bands have a higher concentration of DNA. This helps the observer/researcher to guide the exact band which is under observation. This can help to find out which size of DNA is above and below the reference band. The concentration of all the bands in the DNA ladder is generally provided by the manufacturer as ng of that particular band/1.0 µg of total DNA ladder or ng/5µl of DNA ladder (Fig). As stated in the question the in size of DNA standard decreases from top to bottom and the brightness of the stained gel decreases proportionately. Sometimes, the lower bands in the gel are very faint they are hardly visible. In that case, counting the number of bands may give a wrong impression on the size of DNA. The reference band helps there to identify the correct size.