Double-Stranded DNA Quantitation

Characterization of PicoGreen Reagent and Development of a Fluorescence-Based Solution Assay for Double-Stranded DNA Quantitation

A sensitive assay for detecting double-stranded (ds) DNA in solution is described. This assay employs a new dye, PicoGreen dsDNA quantitation reagent, which becomes intensely fluorescent upon binding nucleic acids. The brightness of this reagent is due to its high quantum yield (0.5, bound to ds calf thymus DNA) and large molar extinction coefficient (70,000 cm⁻¹m⁻¹). The fluorescence enhancement of this dye upon binding dsDNA is >1000-fold, with excitation and emission maxima near those of fluorescein. Unlike Hoechst 33258, PicoGreen reagent fluorescence intensity was the same upon binding to poly(dA)·poly(dT) and poly(dG)·poly(dC) homopolymers. The PicoGreen assay allowed the detection of 25 pg/ml dsDNA, surpassing the sensitivity achieved with Hoechst 33258 by 400-fold. The linear concentration range for DNA quantitation extended over four orders of magnitude—25 pg/ml to 1 μg/ml—with a single dye concentration. Assay linearity was maintained even in the presence of salts, proteins, poly(ethylene glycol), urea, chloroform, ethanol, and agarose; some ionic detergents and heparin interfered. Linear DNAs yielded slightly brighter signals than supercoiled plasmids. Finally, the assay showed greater dsDNA:RNA selectivity than Hoechst 33258 in low ionic strength buffer and better dsDNA:single-stranded DNA selectivity in 1mNaCl.

Biochemical techniques

What is Biochemistry?

Biochemistry

Biochemistry is the study of chemical processes associated with living organisms. Biochemists use concepts of biology, chemistry, physics, mathematics, microbiology, and genetics to unravel the complex puzzles of life. Biochemical techniques are used in clinical diagnosis of infectious diseases, genetic disorders, and cancer; as well as in many forms of research to improve the quality of our lives.

Nutrition

Nutrition is the science that studies the means by which we take in and utilize food. Nutritionists study the metabolism of foods, i.e. how foods are converted and used by the body. They study the need for vitamins and trace elements in all stages of human development from pre-term infants to the elderly.

Dietetics

Dietetics is the application of nutrition to diet planning for individuals, groups and populations. Dietitians are uniquely trained to advise you on food, diet and nutrition. Only a dietitian holds a professional qualification and certain positions (such as food-service directors in hospitals) are restricted to holders of this qualification.

Analysis

Citric acid Analysis


Citric acid was analysed, using pyridine–acetic anhydride method as reported by Marrier and Boulet (1958). One ml of the diluted culture filtrate along with 1.30 ml of pyridine was added in the test tube and swirled briskly. Then 5.70 ml of acetic anhydride was added in the test tube. The test tube was placed in a water bath at 32 ± 0.25 °C for 30 min. The optical density was measured on a spectrophotometer (405 nm) and citric acid contents of the sample were estimated by comparing it with standerds (run parallel, replacing 1.0 ml of the culture filtrate with distilled water).

Composition of Medium For optimization of pH for SSF of molasses using corn cobs as carrier substrate for citric acid production by A. niger
 
Substrate corn cobs
S.no Substrate (g) 10%molasses solution (ml) pH


1 5 15 3

2 5 15 4

3 5 15 5

4 5 15 6

5 5 15 7


Composition of mdium for optimization of inoculum size for citric acid production by A. niger in SSF of molasses medium.
 
Substrate corn cobs
 
S.no Substrate (g) 10%molasses solution (ml) pH Inoculum size


(ml)

1             5          15             6                                 3

2 5 15 6 4

3 5 15 6 5

4 5 15 6 6

5 5 15 6 7

Composition of medium for optimization of temperature


Composition of medium for optimization of nitrogen sources

Substrate corn cobs


S.no

Nitrogen Sources w/w 1%

1 Ammonium sulphate

2 Corn steep liqour

3 Urea

4 Peptone

5 Yeast extract


PH, 6


Inoculum size, 5ml.

Temperature, 35oC.









Substrate corn cobs

S.no pH Inoculum size (ml) Temprature oC








1                 6              3           25

2              6            4                30

3 6 5 35

4 6 6 40

5 6 7 45