Gen5 version required: 1.0 or higher
Basis for the Assay:
Propidium iodide (PI) binds to DNA by intercalating between bases with little or no sequence specificity. Propidium Iodide stain is weakly fluorescent, but when bound to DNA molecules it increases in fluorescent intensity approximately 20-30 fold. 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 485/20 excitation filter and a 645/40 emission filter.
The experiment data file contains all of the fluorescence determinations for a Propidium Iodide, dsDNA determination.
Plate configuration assumes that samples will be run in duplicate. Included is a dsDNA standard curve (0 to 2.5 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.