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#EtBr

2 public questions tagged with this topic.

What is the emission wavelength of EtBr when bound to DNA?

Ethidium bromide is a fluorescent intercalator whose photophysics change dramatically upon DNA binding. Free EtBr in aqueous solution emits weakly, but when intercalated between base pairs, hydrophobic environment shields it and fluorescence quantum yield increases about 20-fold. Upon excitation with UV around 302 nm or 365 nm, the DNA-EtBr complex emits orange-red fluorescence with emission maximum near 590 nm. This property enables visualization of DNA bands in agarose gels under UV transilluminator. Shorter wavelengths like 260 nm or 400 nm are absorption peaks, not emission maxima.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

Which of the following is used to quantify DNA using fluorescence?

Fluorescent quantification of DNA employs DNA-binding fluorochromes for sensitivity beyond UV absorbance. SYBR Green I is a cyanine dye that exhibits extremely low fluorescence when free but over 1000-fold enhancement upon intercalation into minor groove of double-stranded DNA. This allows specific quantitation of dsDNA even with contaminants present. Coomassie Blue stains proteins, DAPI binds AT-rich dsDNA but is used mainly for microscopy, and TRIS is a buffer. SYBR Green is standard for real-time PCR quantification and fluorometric assays with high specificity and linearity.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.