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.
Step 2: Launch SoftMax Pro software and open a new file. (See Figure 1.)
Step 3: Click Set Up. The second screen will appear. (See Figure 2.) First, choose the read type as Fluorescence, then go to each option in the left panel of the screen and set up all the appropriate parameters for the assay: wavelength (EX 480, EM 520 and cutoff at 515), sensitivity of 30 (the higher the number, the higher precision you will get, but it will take longer to read the plate), assay plate type, wells to read, etc.
Step 4: Create a template of the assay showing where the plate blank and sample wells will be located on the microplate. (See Figure 3.)
Prepare the assay
The method for this assay follows the instructions in the product information sheet for OliGreen ssDNA Quantitation Reagent from Molecular Probes, except the assay volume is proportionately reduced from 2.0 ml to 200 l to fit the 96-well microplate format.
Step 1: Prepare 1x TE (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) buffer by diluting the concentrated buffer from the kit 20-fold with distilled DNase-free water, as required by Molecular Probes.
Step 2: Prepare a 2 g/ml and 20 ng/ml solution of oligonucleotide solution from 100 g/ml stock (from the kit) in 1x TE buffer.
Step 3: Dilute 2 g/ml and 20 ng/ml stock solutions. Make a series dilution, as in Table 1, with 1x TE buffer. Include a 0 ng/ml concentration sample to be used as the blank for the plate. The plate blanks RFU values should be subtracted from the RFUs of all samples of the plate.
Step 4: Prepare a working solution of OliGreen reagent immediately prior to the experiment by diluting the concentrated stock 1:200 in 1X TE buffer. Protect it from light by covering with aluminum foil in accordance with Molecular Probes recommendations.
Step 5: Mix the appropriate oligonucleotide solution with OliGreen working solution into eleven brown (for protection from light) eppendorf tubes as described in the following table:
Step 6: Add 100 L of each concentration of oligonucleotide to the wells of the microplate in triplicate. Next add 100 l of working stock of the OliGreen reagent to each sample-containing well in the microplate.
Note: protect the plate from light at all times after adding the OliGreen reagent.
Read the microplate
Step 1: Place the microplate in the reader.
Step 2: Incubate the plate in the instrument for 5 minutes prior to reading the assay.
Step 3: Click the softwares Read button. The spectrofluorometer will read the plate and the relative fluorescence units will be displayed in the Plate section.
Analyze the data
Step 1: After the microplate has been read, the RFUs will be displayed in the Plate section. The data will be analyzed in the Group Tables that you created while setting up the template (see Step 5 of Set up the instrument and software on page 2). For an example of a Group Table see the Results section of this document.
Step 2: Plot the mean RFUs of the samples versus the Oligomer concentrations. (See Figure 4.)
Step 3: Choose the appropriate curve fit from the drop-down Curve Fit menu in the Graph sections tool bar. When plotting a standard curve over the entire dynamic range of the assay use the log-log curve fit. When plotting a smaller portion of the standard curve use a linear curve fit.
The plate was read at several d ifferent excitation/emission wavelength combinations and the data was analyzed. (See Figure 7.)
The following standard curve was obtained using Molecular Probes OliGreen reagent with a random sequence 35-Mer oligonucleotide. (See Figure 5.)
Molecular Probes describes a lower limit of detection of 1 ng/ml when run in a fluorescence microplate reader. The data we have collected confirms this lower limit of detection. The low end of the standard curve is displayed in Figure 6.
Using either excitation/emission at 495/525 or 490/520 is not as good as 480/525 in terms of the noise and signal dynamic range. (See Figure 7.) Identical data and curves were generated from Gemini EM and SpectraMax M2 but are not shown here.
The OliGreen assay from Molecular Probes, when run on a Gemini XS or Gemini EM fluorescence microplate reader with SoftMax Pro software, is a quick, sensitive detection method for single-stranded DNA. The analysis capabilities of SoftMax Pro provide quantitation in an easy-to-read, user-customizable report format. The Gemini XS, Gemini EM and SpectraMax M2 microplate readers provide the flexibility for optimizing the wavelength to ensure optimal read outs.
Molecular Probes Data Sheet MP 7582 02/08/96: OliGreen Oligonucleotides Quantitation Reagent (O-7582) for single-stranded DNA.