Hoechst 33258 (High Concentrations)
November 27, 2012Download
Related Products: Gen5 for Detection
Gen5 version required: 1.0 or higher
Basis for the Assay:
Bisbenzimide stain, also known as Hoechst 33258, binds to dsDNA molecules and markedly increases in fluorescence when it does so. The dye, weakly fluorescent itself in solution, binds specifically to the A-T base pairs in dsDNA resulting in an increase in fluorescence and a shift in the emission maximum from 500 to 460 nm. In the presence of high salt (2M NaCl) the stain is specific for dsDNA, with little to no interference from ssDNA or RNA. The fluorescence intensity is directly proportional to the total nucleic acid concentration present in the sample.
The protocol calls for a fluorescence measurement using a 360/40 excitation filter and a 460/40 emission filter.
The experiment data file contains all of the fluorescence determinations for a Hoechst 33258 stain dsDNA determination.
Plate configuration assumes that samples will be run in duplicate. Included is a dsDNA standard curve (0 to 20 μg/ml). The plate includes 7 standard concentrations, two blank wells, but no controls wells. Thus, 40 samples can be run on a microplate in duplicate, in addition to the standard curve.
The report is configured to provide the (1) raw fluorescence, (2) blanked fluorescence, (3) standard curve, and (4) calculated dsDNA concentrations and statistics of unknown samples.