用于DNA定量的 Hoechst33258染料在Picofluor便携式荧光计上的应用

DNA quantitation is a necessary step for many life science research protocols. Common DNA techniques, such as sequencing, cDNA synthesis and cloning, RNA transcription and transfection, all benefit from a defined template concentration. An inaccurate estimation of the amount of DNA template may cause these techniques to fail.

DNA concentration is often measured by UV absorbance at 260 nm (1A260 = 50 µg/mL) in a 1 cm path length cuvette. This method, however, is not as accurate as DNA quantitation using fluorescent dyes. The Picofluor can be used for DNA quantitation along with Hoechst 33258, a bisbenzimide DNA intercalator that excites in the near UV (350 nm) and emits in the blue region (450 nm). Hoechst 33258 binds to the AT rich regions of double stranded DNA and exhibits enhanced fluorescence under high ionic strength conditions. Sensitivity of the Hoechst 33258 assay in combination with the Picofluor is approximately 10 ng/mL DNA. The linear dynamic range extends over 3 orders of magnitude from 10 ng/mL to 1 µg/mL DNA.