By Yan Zhang., Molecular Devices Corporation, 1311 Orleans Dr., Sunnyvale, CA 94089.
This application note describes how to use the OliGreen Quantitation Kit from Molecular Probes in the Gemini XS, Gemini EM and SpectraMax M2 microplate readers with SoftMax Pro software from Molecular Devices. Single-stranded DNA and oligonucleotides are traditionally measured at 260 nm for absorbance. OliGreen is much more sensitive and specific than traditional photometer methods. The dynamic range of this assay in microplate format is 0.05 ng/ml to 1 g/ml read on Gemini XS. The data presented in this application note confirms the dynamic range and lower detection limit described by Molecular Probes in their application note (MP 07582).
The OliGreen spectra is: excitation/emission: 500/520 nm as indicated in the product insert from Molecular Probes. However, they suggest using excitation/emission wavelengths of 480/520 nm. Because the dual monochromators in Gemini XS allow the selection of any wavelength in 1 nm increments, the assay plate was read with several different excitation/emission wavelengths for optimal dynamic range of the signal. The optimal excitation/emission wavelength used on Gemini XS: 480/520 and cutoff at 515 nm.
OliGreen Oligonucleotide Quantitation Reagent from Molecular Probes (Cat.# O-7582)
Oligonucleotide standard: an M13 sequencing primer from Molecular Probes (Cat.# O-11492)
Black 96-well plate
Brown eppendorf tubes
Spectrofluorometer (Gemini XS, Gemini EM or SpectraMax M2)
SoftMax Pro software (version 4.5 or later)
Set up the instrument and software
Step 1: Turn on the spectrofluorometer.