PicoGmeter, a custom-made fluorometer for the quantification of dsDNA by PicoGreen fluorescence.
The accurate determination of DNA concentration is essential for many processes in molecular biology and physiology and includes both gel- and cuvette-based methods. The recently introduced fluorescent dye, PicoGreen, has several advantages over other methods because it is sensitive and specific for double-stranded DNA (dsDNA). The dye is excited at 480 nm and emits at 520 nm when bound to dsDNA. This report describes the construction and use of PicoGmeter, a simple, inexpensive, fixed-wavelength fluorometer suitable for measuring PicoGreen fluorescence. PicoGmeter employs a blue light emitting diode (LED) for excitation and a photodiode to measure fluorescence. When compared to a commercially available instrument, PTI DeltaScan, the PicoGmeter performed admirably. Calibration curves for both instruments were superimposeable. Moreover, there was no significant difference between concentrations of DNA estimated by both instruments. A Bland and Altman analysis revealed that the PicoGmeter was equivalent to the PTI DeltaScan for estimating dsDNA concentration by the PicoGreen method. This simple, inexpensive, battery-operated fluorometer will allow investigators to employ the PicoGreen method without incurring the cost of purchasing a spectrofluorometer.
Schofield
Geoffrey G
GG
Department of Physiology, Tulane University Health Sciences Center, New Orleans, LA 70112, USA. solar@tulane.edu
D004247:DNA / Q000032:analysis*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005470:Fluorometry / Q000295:instrumentation* / Q000379:methods; D008832:Microchemistry / Q000295:instrumentation* / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D004247:DNA