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感謝參與，應(yīng)助指數(shù) +1
王壽梅: 金幣+2 2012-03-22 14:32:58
感謝參與，應(yīng)助指數(shù) +1
王壽梅: 金幣+2 2012-03-22 14:32:58
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Spectrophotometry and fluorometry are commonly used to measure DNA concentration. Spectrophotometrycan be used to measure microgram quantities of pure DNA samples (i.e., DNA that is not contaminated by proteins, phenol, agarose, or RNA). Fluorometry is more sensitive, allowing measurement of nanogram quantities of DNA, and furthermore the use of Hoechst 33258 dye allows specific analysis of DNA. 1。Spectrophotometry DNA concentration can be determined by measuring the absorbance at 260 nm (A260) in a spectrophotometer using a quartz cuvette. For greatest accuracy, readings should be between 0.1 and 1.0. An absorbance of 1 unit at 260 nm corresponds to 50 μg genomic DNA per ml (A260 =1 ⇒ 50 μg/ml)*. This relation is valid only for measurements made at neutral pH, therefore, samples should be diluted in a low-salt buffer with neutral pH (e.g., Tris·Cl, pH 7.0). An example of the calculation involved in nucleic acid quantification when using a spectrophotometer is provided in “Spectrophotometric Measurement of Nucleic Acid Concentration” 1）If you will use more than one cuvette to measure multiple samples, the cuvettes must be matched. 2）Spectrophotometric measurements do not differentiate between DNA and RNA, so RNA contamination can lead to overestimation of DNA concentration. 3）Phenol has an absorbance maximum of 270–275 nm, which is close to that of DNA. Phenol contamination mimics both higher yields and higher purity, because of an upward shift in the A260 value. 2.Fluorometry Fluorometry allows specific and sensitive measurement of DNA concentration by use of the fluorochrome Hoechst 33258, which shows increased emission at 458 nm when bound to DNA. This dye has little affinity for RNA, allowing accurate quantification of DNA samples that are contaminated with RNA.DNA standards and samples are mixed with Hoechst 33258 and measured in glass or acrylic cuvettes using a scanning fluorescence spectrophotometer or a dedicated filter fluorometer set at an excitation wavelength of 365 nm and an emission wavelength of 460 nm. The sample measurements are then compared to the standards to determine DNA concentration. 1）As Hoechst 33258 preferentially binds AT-rich DNA, use standards with a similar base composition to the sample DNA. |
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