The ambdaDNA standard provided in the kit can be prepared by diluting it 50 times with TE to make a 2 μg/mL solution. Note: In some cases, it may be necessary to use a DNA type similar to the DNA to be tested to prepare a standard curve-high concentration range can be prepared from 1 ng/mL to 1 μg/mL, and low concentration range can be from 25 pg/ mL to 25 ng/mL. For the high-concentration range standard, dilute according to the dilution scheme in Table 2. For the low-concentration range standard, prepare a 50 ng/mL solution by diluting a 2 μg/mL solution 40 times. Note: In this application document, a series of calibrations ranging from 1 μg/mL to 50 pg/mL are used. -Use a pipette to transfer 100 μL of each well standard to a black 96-well microplate, preferably 3 replicates. Make sure to add TE (no DNA) to the well as a blank well. -Add 100 μL of Quant-iT PicoGreen reagent aqueous working solution per well. Mix by vortexing or shaking at room temperature in the dark for 2 to 5 minutes. Reading microplates-Make sure that the purple plate adapter (if required by your plate reader) is placed in the plate holder of the microplate reader. -Click the Read button in SoftMax Pro software. The instrument will read the board and the relative fluorescence unit will be displayed in the board section of the program. Analysis data-After reading the microplate, the relative fluorescence units (RFUs) will be displayed in the plate section. The data in the group 10ul Filter Tip set by the template creation will be analyzed. For the data in a representative set of tables, see the results section of Table 4. -The assigned standard in the template will be automatically fitted into a curve in the standard curve block in the program (and displayed in the standard group table). The default setting is linear curve fitting, but loglog curve fitting can be used when fitting a wide range of scales. You can select curve fitting in the curve fitting drop-down menu in the graph area. Results The Quant-iT PicoGreen dsDNA experiment and the SpectraMax? M5 microplate reader were used to determine the DNA standard concentration from 1 μg/mL to 50 pg/mL
