Hoechst 33258 (Low Concentrations)

Gen5 version required: 1.0 or higher


Basis for the Assay:

Bisbenzidine stain, also known as Hoechst 33258, binds to dsDNA molecules and markedly increases in fluorescence when it does so. 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:

Plate configuration assumes that samples will be run in duplicate. Included is a dsDNA standard curve (0 to 400 ng/ml). The plate includes 8 standard concentrations, but no blank or control wells. Thus, 40 samples can be run on a microplate in duplicate, in addition to the standard curve.

Report:

The report is configured to provide the (1) raw fluorescence, (2) standard curve, and (3) calculated dsDNA concentrations and statistics of unknown samples.