signal. The optimal excitation/emissionwavelength used on Gemini XS: 480/520 andcutoff at 515 nm.

MATERIALS> 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 EMor SpectraMax M2)

> SoftMax Pro software (version 4.5 or later)

METHODS

Set up the instrument and softwareStep 1: Turn on the spectrofluorometer.

Step 2: Launch SoftMax Pro software and open anew file. (See Figure 1.)

Step 3: Click “Set Up.” The second screen willappear. (See Figure 2.) First, choose the readtype as Fluorescence, then go to each optionin the left panel of the screen and set up allthe appropriate parameters for the assay:wavelength (EX 480, EM 520 and cutoff at515), sensitivity of 30 (the higher thenumber, the higher precision you will get, but

Using the OliGreen Oligonucleotide Quantitation Reagent in theGemini XS, Gemini EM and SpectraMax M2 Microplate Readers

By Yan Zhang., Molecular Devices Corporation, 1311 Orleans Dr., Sunnyvale, CA 94089.

INTRODUCTION

This application note describes how to use theOliGreen® Quantitation Kit from Molecular Probesin the Gemini XS, Gemini EM and SpectraMax®

M2 microplate readers with SoftMax® Pro softwarefrom Molecular Devices. Single-stranded DNA andoligonucleotides are traditionally measured at 260nm for absorbance. OliGreen is much more sensitiveand specific than traditional photometer methods.The dynamic range of this assay in microplateformat is 0.05 ng/ml to 1 µg/ml read on GeminiXS. The data presented in this application noteconfirms the dynamic range and lower detectionlimit described by Molecular Probes in theirapplication note (MP 07582).

The OliGreen spectra is: excitation/emission:500/520 nm as indicated in the product insertfrom Molecular Probes. However, they suggestusing excitation/emission wavelengths of480/520 nm. Because the dual monochromatorsin Gemini XS allow the selection of anywavelength in 1 nm increments, the assay platewas read with several different excitation/emissionwavelengths for optimal dynamic range of the

APPLICATION NOTE #21

The initial blank experiment template in SoftMax Pro.Using SoftMax Pro, you can select assay parameters such asassay type, read type and wavelengths.

assay parameters set-up (figure 2)initial experiment set-up (figure 1)

it will take longer to read the plate), assayplate type, wells to read, etc.

Step 4: Create a template of the assay showingwhere the plate blank and sample wells willbe located on the microplate. (See Figure 3.)

Prepare the assayThe method for this assay follows the instructions inthe product information sheet for OliGreen ssDNAQuantitation Reagent from Molecular Probes, exceptthe assay volume is proportionately reduced from 2.0ml to 200 µl to fit the 96-well microplate format.

Step 1: Prepare 1x TE (10 mM Tris-HCl, 1 mMEDTA, pH 7.5) buffer by diluting theconcentrated buffer from the kit 20-fold withdistilled DNase-free water, as required byMolecular Probes.

Step 2: Prepare a 2 µg/ml and 20 ng/ml solutionof oligonucleotide solution from 100 µg/mlstock (from the kit) in 1x TE buffer.

Step 3: Dilute 2 µg/ml and 20 ng/ml stocksolutions. Make a series dilution, as in Table 1,with 1x TE buffer. Include a 0 ng/mlconcentration sample to be used as the blankfor the plate. The plate blank’s RFU valuesshould be subtracted from the RFUs of allsamples of the plate.

USING OLIGREEN REAGENT IN GEMINI XS, GEMINI EM AND SPECTRAMAX M2 MICROPLATE READERS

Step 6: Add 100 µL of each concentration ofoligonucleotide to the wells of the microplatein triplicate. Next add 100 µl of workingstock of the OliGreen reagent to each sample-containing well in the microplate.

Note: protect the plate from light at all times afteradding the OliGreen reagent.

Read the microplateStep 1: Place the microplate in the reader.

Step 2: Incubate the plate in the instrument for5 minutes prior to reading the assay.

Step 3: Click the software’s Read button. Thespectrofluorometer will read the plate andthe relative fluorescence units will bedisplayed in the Plate section.

Analyze the dataStep 1: After the microplate has been read, the

RFUs will be displayed in the Plate section.The data will be analyzed in the GroupTables that you created while setting up thetemplate (see Step 5 of “Set up theinstrument and software” on page 2). For anexample of a Group Table see the Resultssection of this document.

Group Table indicating blank and sample wells. Plotting the standard curve.

group table set-up (figure 3) standard curve set-up (figure 4)

Step 4: Prepare a working solution of OliGreenreagent immediately prior to the experimentby diluting the concentrated stock 1:200 in1X TE buffer. Protect it from light bycovering with aluminum foil in accordancewith Molecular Probes’ recommendations.

Step 5: Mix the appropriate oligonucleotide solutionwith OliGreen working solution into elevenbrown (for protection from light) eppendorftubes as described in the following table:

Table 1. Oligonucleotide Dilution Oligo-

nucleotideConcentration

(ng/ml)

OligonucleotideVolume

1x TEBuffer

Solution(µl)

Final assayOligonucleotide Concentration

(ng/ml)

0 (blank) 0 1000 0 (blank)

0.1 5 µl of 20 ng/ml 995 0.05

0.4 20 µl of 20 ng/ml 980 0.2

1 50 µl of 20 ng/ml 950 0.5

2 100 µl of 20 ng/ml 900 1

4 200 µl of 20 ng/ml 800 2

20 10 µl of 2 ng/ml 990 10

40 20 µl of 2 ng/ml 980 20

200 100 µl of 2 ng/ml 900 100

400 200 µl of 2 ng/ml 800 200

2000 1000 µl of 2 ng/ml 0 1000

APPLICATION NOTE #21

Step 2: Plot the mean RFUs of the samples versusthe Oligomer concentrations. (See Figure 4.)

Step 3: Choose the appropriate curve fit fromthe drop-down Curve Fit menu in the Graphsection’s tool bar. When plotting a standardcurve over the entire dynamic range of theassay use the log-log curve fit. When plottinga smaller portion of the standard curve use alinear curve fit.

Wavelength optimizationThe plate was read at several differentexcitation/emission wavelength combinationsand the data was analyzed. (See Figure 7.)

RESULTS

The following standard curve was obtained usingMolecular Probes’ OliGreen reagent with arandom sequence 35-Mer oligonucleotide. (SeeFigure 5.)

Molecular Probes describes a lower limit ofdetection of 1 ng/ml when run in a fluorescencemicroplate reader. The data we have collectedconfirms this lower limit of detection. The lowend of the standard curve is displayed in Figure 6.

Using either excitation/emission at 495/525 or490/520 is not as good as 480/525 in terms ofthe noise and signal dynamic range. (See Figure 7.)Identical data and curves were generated fromGemini EM and SpectraMax M2 but are notshown here.

CONCLUSIONS

The OliGreen assay from Molecular Probes,when run on a Gemini XS or Gemini EMfluorescence microplate reader with SoftMax Prosoftware, is a quick, sensitive detection methodfor single-stranded DNA. The analysiscapabilities of SoftMax Pro provide quantitationin an easy-to-read, user-customizable reportformat. The Gemini XS, Gemini EM andSpectraMax M2 microplate readers provide theflexibility for optimizing the wavelength toensure optimal read outs.

REFERENCES

Molecular Probes Data Sheet MP 7582 02/08/96:OliGreen Oligonucleotides Quantitation Reagent(O-7582) for single-stranded DNA.

Standard curve obtained using OliGreen and

24-Mer sequencing primer (log-log curve fit).

Detail of the lower end of the standard curve

shown in Figure 5. Comparing the standard curves for different wavelengths.

0.01 0.1 1 10 100 10000.01

0.1

1

10

100

1000

10,000

RFU

s

oligomer (ng/ml)0 0.5 1.5 21

0

1

2

3

RFU

s

oligomer (ng/ml)0.01 0.1 1 10 100 1000

0.01

0.1

1

10

EX/EM 495/525 (495/525: conc. vs. mean value) 0.615 0.833 0.975 Log(y)= A+B * Log(x) A B C

EX/EM 490/520 (490/520: conc. vs. mean value) 0.395 0.830 0.953

EX/EM 480/525 (480/525: conc. vs. mean value) 0.254 0.927 0.995

100

1000

10,000

RFU

s

oligomer (ng/ml)

OliGreen standard curve (figure 5) standard curve low end detail (figure 6) wavelength optimization (figure 7)

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