The overall aim of the following experiment is to determine if a potentially heterogeneous sample of biomolecules contains nucleic acid and or reducing sugars. And if so, distinguish between RNA and DNA based on the differing reactivity of their sugar moieties. This is achieved by first applying the Benedict assay to determine if the biomolecular mixture contains free reducing sugars.
As a second step files, arsen or assay is used, which establishes whether or not any sugar components contain pento rings. Next dishes Diphenyl amine reagent is used to determine whether the pento sugar is a deoxyribose as in DNA or not as in RNA. The results show the type of biopolymer based on readily distinguished color products formed by the differential reactivities of sugar-based components in the sample, which can be analyzed against standard curves using spectro photometric measurements.
The main advantage of this technique over existing methods such as electrophoresis using fluorescent dyes, pico green, or cyber gold, is that there is no limitation based on size, charge, or sugar component, and it is efficient in time and cost To assay for reducing sugars. Prepare a suitable volume of six x Benedict's reagent composed of 940 millimolar anhydrous, sodium carbonate 588 millimolar, sodium citrate dihydrate, and 68 millimolar copper. Two sulfate penta hydrate.
For each sample to be assay, add 100 microliters of the six x Benedict reagent to 1.5 milliliter micro centrifuge tube. Add between 10 to 500 microliters of sample to each tube and add double distilled water to bring the final volume to 600 microliters vortex or pipette. To mix the solution.
Incubate the samples in a boiling water bath for 20 minutes, and then allow them to cool for 10 minutes before centrifusion at around 10, 000 RPM for five minutes. To sediment any particulate material, transfer the supinate into a clean vete after blanking the UV vs. Spectrophotometer with water at 475 nanometers.
Measure the absorbence of the sample to assay for pento sugars. Prepare fresh two x BALs reagent with 24.2 millimolar, six molar hydrogen chloride and 0.025 Weight per volume ferric chloride hexahydrate according to the text protocol. For each sample, add 500 microliters of biles reagent to a micro centrifuge tube.
Add between 10 and 500 microliters of sample to each tube and add double distilled water to bring the final volume to one milliliter. A mix. Boil the samples for 20 minutes, then cool it room temperature for 10 minutes after spinning down the sample to sediment the particulate material, measure the absorbance at 660 nanometers using water as a blank.
Prepare two x dishes diphenyl amine reagent composed of 60 millimolar diphenyl amine, seven molar glacial acetic acid, 179 millimolar sulfur acid, and 62%volume per volume.Ethanol. For each reaction, combine 500 microliters of reagent, the sample and double distilled water to bring the total volume to one milliliter. After boiling samples for 20 minutes, cooling up room temperature and spinning, measure the absorbance at 600 nanometers.
Shown here her representative qualitative data for the Benedicts Biles arsenal and dishes, diphenyl amine assays for known reference compounds. The left panels show both positive and negative controls, and the right panels display the visible detection range of the assays. This panel illustrates the robustness of the assays by showing the dish's reaction with samples of varying heterogeneity.
For example, DNA with RNA or DNA with protein. Note that the positive results of the Dish's assay is preserved for DNA containing samples even in the presence of contaminating RNA or protein. The dilution ranges in the previous samples vary because each reaction has a distinct visual detection limit.
Depending on the type of sugar being assayed, spectra photometric rather than visual detection, can be used to improve the measurement ranges as shown in this standard curve. For the Benedict assay, Once mastered, this technique can be done in under 30 minutes if performed properly.
