#dna quantification

Hello everyone,      

Today I am discussing a different method for quantification of DNA ie Quantifying DNA in Solution with PicoGreen

  I have covered similar topics earlier....

DNA QUANTIFICATION USING MINIGELS

Estimating the concentration of DNA by Fluorometry using Hoechst 33258

PicoGreen dsDNAQuantitation Reagent is an ultra-sensitive fluorescent nucleic acid stain for quantitating double-stranded DNA (dsDNA) in molecular biological procedures such as cDNA synthesis for library production and DNA fragment purification for subcloning, as well as diagnostic applications, such as quantitating DNA amplification products and primer extension assays.

The PicoGreen assay allowed the detection of 25 pg/ml dsDNA , surpassing the sensitivity achieved with Hoechst 33258 by 400-fold. The assay is most quickly and easily performed using a handheld fluorometer.

The standard PicoGreen assay protocol is also simpler than the Hoechst 33258 method, because a single concentration of the PicoGreen Reagent allows detection over the full dynamic range of the assay.Unlike hoechst – based assay, assay using a single concentration of picogreen are linear over four orders of magnitude in DNA concentration from 1ng/ml to 1 µg/mL.

  PRINCIPLE:-

PicoGreen  dsDNA Quantitation Reagent is an ultra-sensitive fluorescent nucleic acid stain for quantitating double-stranded DNA (dsDNA) in molecular biology procedures.The brightness of this reagent is due to its high quantum yield and large molar extinction coefficient. These procedures include cDNA synthesis for library production, DNA fragment purification for sub cloning, and diagnostic applications, such as quantitating DNA amplification products,  and primer extension assays.

  REAGENTS

       Picogreen dsDNA quantitation reagent

       Reference DNA (eg calf thymus DNA) at concentration of 2 µg/mL  in TE(pH 7.5)

        TE (pH 7.5)

To read more http://microamaze.blogspot.in/2016/01/quantifying-dna-in-solution-with.html 

Other methods for DNA Quantification DNA QUANTIFICATION USING MINIGELS Estimating the concentration of DNA by Fluorometry using Hoechst 33258 Extraction and Purification of Plasmid DNA by Alkaline Lysis MethodEstimation of DNA by Diphenylamine method

With love

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

DNA Quantification Using Ethidium bromide to estimate the amount of DNA in bands after Electrophoresis through Minigels

INTRODUCTION :-

It is a Rapid method. Both measure quantity and analyse its physical state. Even less than 5ng of DNA can be detected. Amount of DNA  in an unknown sample is estimated by comparing intensity of image captured , with image obtained from set of standards

PRINCIPLE:-

Ethidium bromide may be a mutagen, a carcinogen, or a teratogen, although this depends on the organism exposed and the circumstances of exposure. Ethidium bromide is an intercalating agent commonly used as a fluorescent tag (nucleic acid stain) in molecular biology laboratories for techniques such as agarose gel electrophoresis.

When exposed to ultraviolet light, it will fluoresce with an orange colour, intensifying almost 20-fold after binding to DNA. Ethidium bromide possesses UV absorbance maxima at 300 and 360 nm . Additionally, it can absorb energy from nucleotides excited by absorbance of 260 nm radiation.Ethidium re-emits this energy as yellow/orange light centered at 590 nm. The fluorescence of ethidium bromide in aqueous solution is significantly lower than that of the interchelated dye.

REAGENTS:-

•      Agarose minigels (0.6%) containing ethidium bromide (0.5 µg/mL  ) :-These are small size gells with dimensions 8X10cm and volume of agarose required is 30 - 50ml

•      DNA standards.

•      Electrophoresis buffer (1xTAE)

•      Gel loading buffer IV

•      mgcl2

To read more http://microamaze.blogspot.in/2015/11/dna-quantification.html

With love