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当前位置: 首页 > 文献中心 > DETERMINATION OF DNA CONCENTRATION AND PURITY

DETERMINATION OF DNA CONCENTRATION AND PURITY

来源:贝斯特试剂    发布时间:2013-04-14    浏览次数:3771

 

  Different methods have been developed to determine or estimate the purity and concentration of DNA, such as UV spectrophotometry (1), diphenylamine assay (1, 2), restriction enzyme digestion and gel electrophoresis. Each procedure has advantages and disadvantages. For example, UV spectrophotometry is quite simple for the determination of DNA concentration and purity. However, it but can not be used to estimate the DNA concentration if the DNA sample is contaminated with RNA and some degraded DNA. Moreover, when the DNA sample is contaminated with some organic materials and/or ions, which will inhibit enzymatic reactions (such as restriction digestion), this method can not give any information about the contamination. In these cases, the restriction digestion and gel electrophoresis method can be used to distinguish DNA, degraded DNA, RNA and different contaminants and estimate the DNA concentration if a standard marker is used.
  The diphenylamine reagent can be used in a quantitative colorometric assay specific for thymidine residues in crude extracts. The specificity of the reaction for thymidine bases means that the assay can discriminate easily between RNA and DNA, and is much more accurate than reading optical density of the nucleic acid solution at 260 nm. The difference in the nucleic acid content determined by OD260 and the diphenylamine test will obviously give an indication of the amount of RNA in the preparation. However, this procedure has more steps than others and requires micro cuvettes to read the OD600  value, otherwise, large amount of DNA sample is needed for the test (There is a linear relationship between OD600 and DNA concentration between the limits of final 5-50 μg /ml of DNA).
  The most common method for the determination of nucleic acid concentrations in nucleic acid solution at room temperature is to measure the absorbance at 260 nm since it is quite simple even though it has some disadvantages described above.