Quantification of DNA nucleic acid concentration can be used to (1) determine the concentration of purified PCR products, (2) determine the concentration of purified digests, and (3) determine the concentration of extracted plasmids.
Operation method
Experiments for the determination of DNA nucleic acid concentration
Principle
Oligonucleotides, single-stranded and double-stranded DNA, and RNA can be quantitatively soluble in buffer, constitute nucleic acids purine bases and pyrimidine bases have conjugated double bonds, so that the nucleoside nuclei and nucleic acids in the ultraviolet light with characteristic absorption spectra, the maximum absorption peak of 260 nm.Narrow-band ultraviolet spectrophotometer, the length of 260 nm.1 cm of the optical diameter of the cuvette.1 absorbance value (1A) is equivalent to 50ug/ ml double helix DNA, 40ug/m1 single helix DNA or RNA), 20 ug/ml oligonucleotide. Prior to testing, the correct program was selected and the volumes of stock and dilution solutions were entered, ergo the blank and sample solutions were tested. However, the experiment was not always smooth. Erratic readings can be a major headache for the experimenter. The higher the sensitivity of the instrument, the greater the drift in absorbance values.
Materials and Instruments
Sample DNA Move 1. Take 10ul of DNA solution, add 600ul of double-distilled water (i.e., dilute 61 times). Caveat Too high a concentration of ions during the test can also lead to drift in the readings, so it is recommended to use a buffer with a certain pH and a low concentration of ions, such as TE, which can stabilize the readings considerably. The dilution concentration of the sample is also a factor that should not be ignored: due to the inevitable presence of some fine particles in the sample, especially nucleic acid samples. The presence of these small particles interferes with the test results. In order to minimize the effect of particles on the test results, it is required that the absorbance value of the nucleic acid is at least greater than 0.1 A, and preferably in the range of 0.1-1.5 A. In this range, the interference of particles is relatively small and the results are stable. From there, it means that the concentration of the sample cannot be too low, or too high (beyond the test range of the photometer). Finally, there are operational factors, such as mixing should be sufficient, otherwise the absorbance value is too low, or even negative; the mixture can not exist air bubbles, the blank liquid without suspension, otherwise the readings drift drastically; must use the same colorimetric cup to test the blank and the sample, otherwise the concentration difference is too large; the conversion factor and the concentration of the sample unit selection of the same; can not be used in the window of the worn out cuvette; the sample's volume must be up to the cuvette requirements of the Minimum volume of the sample must reach the minimum volume required by the colorimetric cup and many other operational matters. Common Problems In addition to the nucleic acid concentration, the spectrophotometer also displays several very important ratios that indicate the purity of the sample, such as the A260/A280 ratio, which is used to evaluate the purity of the sample because the absorption peak of the protein is at 280 nm. For a pure sample, the ratio is greater than 1.8 (DNA) or 2.0 (RNA). If the ratio is lower than 1.8 or 2.0, it indicates the presence of proteins or phenolics.A230 indicates the presence of some contaminants in the sample, such as carbohydrates, peptides, phenols, etc. Pure samples with a ratio of A260/A230 are greater than 2.0.A320 detects turbidity and other interfering factors in the solution. pure samples, A320 is usually 0. For more product details, please visit Aladdin Scientific website.
Double-distilled water
UV Spectrophotometer Colorimetric cups
2. Zero with double-distilled water, and determine the absorbance values at 260nm and 280nm (A260. A280).
3. DNA concentration (ug/ul) = A260x 50x 61/1000
4. DNA purity: the ratio of A260/A280 should be greater than 1.7 or more. If the sample contains impurities such as proteins (absorption peak at 280nm), the ratio drops and the sample should be repurified.