使用Hoechst 33258通过荧光法估算DNA浓度。

Estimating the Concentration of DNA by Fluorometry Using Hoechst 33258.

作者信息

Green Michael R, Sambrook Joseph

出版信息

Cold Spring Harb Protoc. 2017 May 1;2017(5):2017/5/pdb.prot093567. doi: 10.1101/pdb.prot093567.

Abstract

Measuring the concentration of DNA using fluorometry is more sensitive than spectrophotometry, allowing the detection of nanogram quantities of DNA. In this assay, DNA preparations of known and unknown concentrations are incubated with the fluorochrome Hoechst 33258. Absorption values for the unknown sample are compared with those observed for a known dilution series, and the concentration of the unknown sample is estimated by interpolation. The assay can only be used to measure the concentration of DNAs whose sizes exceed ∼1 kb, as Hoechst 33258 binds poorly to smaller DNAs.

摘要

使用荧光法测量DNA浓度比分光光度法更灵敏，能够检测到纳克量的DNA。在该检测中，将已知和未知浓度的DNA制剂与荧光染料Hoechst 33258一起孵育。将未知样品的吸光值与已知稀释系列的吸光值进行比较，并通过插值法估算未知样品的浓度。由于Hoechst 33258与较小的DNA结合不佳，该检测仅可用于测量大小超过约1 kb的DNA的浓度。