Estimating the concentration of DNA by Fluorometry using Hoechst 33258
Hello everyone,Today I am going to write about one of the methods used to determine Concentration of DNA ie estimating the concentration of DNA by Fluorometry using Hoechst 33258. I have already covered some methods to determine concentration of DNA
AGAROSE GEL ELECTROPHORESIS, Estimation of DNA by Diphenylamine method , DNA QUANTIFICATION USING MINIGELS
INTRODUCTION:-
Quantitation of DNA is a prelude to many practices in Molecular Biology. Accurately determining the DNA concentrations of crude chromosomal or purified plasmid DNA samples is not only desirable, but an essential step in quantitative manipulations of DNA. Common techniques that use DNA, such as sequencing, cDNA synthesis and cloning, RNA transcription, transfection, nucleic acid labeling (e.g. Random prime labeling), etc., all benefit from a defined template concentration. Failure to produce results from these techniques can sometimes be attributed to an incorrect estimate of the DNA template used. DNA concentration is measured by UV absorbance at 260 nm (1A260 = 50 µg/mL) in a 1cm path length cuvette.
PRINCIPLE:-
Hoechst 33258 (bisbenzimidazole ) is indol derived stain in free solution has maximum at 356 nm and emission maximum at 492nm . Hoechst 33258 only bind efficiently to small fragments of DNA. Concentration of Hoechst 33258 in reaction mixture should be kept low (5X10-7 M to 2.5X10-6). All DNAs and solutions should be free of ethidium bromide. Hoechst 33258 dye is cell permanent.
Fluorometry assay with Hoechst 33258 do not work at extremes of pH and are affected by both detergents and salts. The dye, weakly fluorescent itself in solution, binds specifically to the A-T base pairs in dsDNA resulting in an increase in fluorescence and a shift in the emission maximum from 500 to 460 nm.
The use of Hoechst 33258 in conjunction with the fluorescence microplate reader offers high specificity, as well as high sensitivity for ds DNA quantitation. The concentration of DNA in unknown sample is estimated from std curve constructed using set of std dna (0-20 µg/mL ) whose base composition is Same as unknown.
REAGENT :-
DNA of known concentration required to construct a std curve
Florometry buffer :- 2MNaCl , 50 mM sodium phosphate (pH7.4). Prepare 500mL and srerlize through a 0.45 filter
High molecular weight DNA solution (100 µg/ml, in TE pH 8.0)
Hoechst 33258 dye (1 mg /ml in water)
Test DNA sample.
EQUIPMENT:-
Low fluorescent background
Black , U shaped ,96- Microfluor B plates
Plate reader
To read more http://microamaze.blogspot.in/2016/01/estimating-concentration-of-dna-by.html
With love
-Dixy