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