Crystal Violet is a popular DNA binding dye. It is widely known that fluorescence emission of Crystal Violet (CV) is significantly enhanced upon titrations with anti-parallel G-quadruplex DNA. Upon exciting CV at 540 nm it gives a characteristic positive maxima around 640 nm. However, we got emission maxima at 580 nm instead of 640 nm both in presence and absence of DNA. The fluorescence intensity is very much close to baseline (I>8) and does not significantly enhance upon DNA titrations.

1. We first dissolved 10 uM CV in water (pH 7.0) from a 1 mM stock dissolved in water (prepared one day before; stored in dark, 4 degree Centigrade). We set the excitation maxima at 540 nm and monitored the spectra from 550 to 700 nm. We got very weak signal (I=2.0) at 580 nm.

2. We titrated glycerol into CV upto 80% Glycerol. The fluorescence intensity is enhanced to I=4.

3. We then freshly prepared the 1 mM stock in 30 % glycerol and reiterated the same steps but observed no change.

4. We did not observe escalation of fluorescence emission upon anti-parallel G-quadruplex (Telomere) titration at 1:1 ratio.

4. We monitored the emission spectra of free and DNA-bound CV at acidic and basic pH. but we observed no change in the quality of the spectra.