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