🧪 MEASUREMENT OF DNA, RNA OR PROTEIN CONCENTRATION | SPECTROPHOTOMETER VS. FLUORESCENCE-BASED METHOD
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2 سال پیش
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Measuring how much DNA, RNA
Measuring how much DNA, RNA or protein you have, via absorbance of light of the molecules in a spectrophotometer, has several advantages. The advantages include speed, small sample volumes and the use of the inherent properties of the DNA, RNA or protein molecule to make the assessment. Notable amongst these advantages is the fact that you do not need any dyes or reagents; you measure direct absorption of light by the biomolecule. Benzene rings in the DNA/RNA molecules and Tryptophan amino acid residues in proteins, allow for this characteristic interaction with ultraviolet (UV) light. The disadvantage of direct detection of DNA, RNA or protein, is that interferring compounds and contaminants such as the reagents used to prepare the biomolecule for analysis, can interfere with its absorption of light, causing over or underestimation of the concentration. Common contaminants include salts, free nucleotides, non-target biomolecules, solvents or detergents. Standard UV absorbance readings are also not sensitive enough to detect low levels of analyte and do not distinguish between RNA vs DNA, quantitating all nucleic acid present in a sample. However, you can differentiate between DNA and RNA by looking at the A260/A280 ratio; For RNA it's expected to be closer to 2.1; while dsDNA will be ~1.85-1.88.
To get away from interfering or contaminating compounds, a specific dye that fluoresces when bound to DNA, RNA or protein may be used. Fluorescent molecules (called fluorophores), when excited by a specific wavelength of ultraviolet light have the ability to emit their own light, at a different (lower energy) wavelength. Using such reagents allow you to avoid interfering or contaminating compounds when measuring concentration.
Using an instrument such as the Qubit, reagents can be purchased as a kit, to perform fluorescence-based measurements of your DNA, RNA or protein. To do this, dilute the fluorescent dye (200x concentrated) to 1x using the reagent diluent. That is, add 1part dye to 199 parts of the diluent. The assay is recommended to be performed in a 200ul volume for each sample. If you have multiple samples, then multiply the number of samples that you want to measure, by 200ul.
For example, if you need 400ul in total for the number of samples that you have to assay, add 2ul of the dye to 398ul of diluent (400ul volume / 200x dye).
Add 2ul of your sample (DNA/RNA ) to 198ul of the diluted dye (if doing 1:100 dilution of your sample). If doing less than 1:100 dilution of the input sample, then revise the ratio of sample to diluted dye.
A 1:100 dilution is ok for the DNA High Sensitivity Kit, but may not be appropriate for the Broad range kit, for instance.
Vortex the tubes (provided in the Qubit reagent kit) containing the dye:sample mix for 3 sec to mix
Incubate Room Temp for 2min before reading the quantity of nucleic acid in the sample. To read your fluorescence-based read out in the Qubit fluorometer, select Assay type, insert the tube, then close the tube before reading the sample.
To get away from interfering or contaminating compounds, a specific dye that fluoresces when bound to DNA, RNA or protein may be used. Fluorescent molecules (called fluorophores), when excited by a specific wavelength of ultraviolet light have the ability to emit their own light, at a different (lower energy) wavelength. Using such reagents allow you to avoid interfering or contaminating compounds when measuring concentration.
Using an instrument such as the Qubit, reagents can be purchased as a kit, to perform fluorescence-based measurements of your DNA, RNA or protein. To do this, dilute the fluorescent dye (200x concentrated) to 1x using the reagent diluent. That is, add 1part dye to 199 parts of the diluent. The assay is recommended to be performed in a 200ul volume for each sample. If you have multiple samples, then multiply the number of samples that you want to measure, by 200ul.
For example, if you need 400ul in total for the number of samples that you have to assay, add 2ul of the dye to 398ul of diluent (400ul volume / 200x dye).
Add 2ul of your sample (DNA/RNA ) to 198ul of the diluted dye (if doing 1:100 dilution of your sample). If doing less than 1:100 dilution of the input sample, then revise the ratio of sample to diluted dye.
A 1:100 dilution is ok for the DNA High Sensitivity Kit, but may not be appropriate for the Broad range kit, for instance.
Vortex the tubes (provided in the Qubit reagent kit) containing the dye:sample mix for 3 sec to mix
Incubate Room Temp for 2min before reading the quantity of nucleic acid in the sample. To read your fluorescence-based read out in the Qubit fluorometer, select Assay type, insert the tube, then close the tube before reading the sample.
2 سال پیش
در تاریخ 1401/09/23 منتشر شده
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