Posted on May 18th, 2016 by UNM CC
M. Fero 11/05 • Adapted from Turner's protocol (website)
RNA: Ribogreen 200x (Invitrogen R-11491) detection reagent. Store dessicated at -20ºC.
Turner Designs TD-360 Fluorometer
RNA or DNA standards (100 ng/µL)
TE pH 8 (DNA), pH 7.5 (RNA)
Fluorometry utilizes fluorescent dyes which specifically bind DNA or RNA. It requires a negative control (to set the zero point on the fluorometer) and a standard of known concentration. The fluorometer shines light on the sample (excitation) and then measues level of fluorescent light being emitted to the side (at a 90º angle) of the excitation light beam. The fluorescent dyes are relatively specific to nucleic acids as opposed to protein and other cellular components. The fluorescence of the dyes increases when they bind nucleic acids. Fluorometry is about 1,000x more sensitive than spectrophotometric absorbance (i.e. measurement of A260) and less susceptible to protein and RNA contamination. However it also does not give a crude measurement of purity (like an A260/A280 ratio) nor does it assure that the DNA or RNA is not degraded (e.g. like size determination by gel electrophoesis). Do not use glass (spectrophotmetry) cuvettes in a fluorometer because the frosted glass on the side of the cuvette interferes with detection of fluorescent light.
|High Range Solution||20 ng - 1 µg||1 - 50 ng|
|Low Range Solution||1 - 50 ng||0.1 - 5 ng|
(High range assay solution)
Note: The procedure for "Low range" assays is identical but it uses the detection reagent diluted 1/10x the cocentrations listed above to minimize background autofluorescence.
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