The main difference is in the concentration of the dsDNA binding dye inside the 200x solution and in dsDNA HS Assay Kit. According to our measurements, the dye concentration in the Pico488 quantification kit is two times higher compared to the original dsDNA HS Assay Kit. This gives our customer a chance to measure even smaller concentrations on the reader using Pico488 DNA quantification reagent when following the original protocol.
So there are two options to follow:
Option 1. When following the original protocol provided for the Q32854 kit by Invitrogen. The range of dsDNA concentration measurements in practice is 1 pg/μL – 10 ng/μL, which can give the UHS (ultra-high sensitivity) to the experiment performed.
Option 2. When diluting the more concentrated Pico488 DNA quantification reagent 1:400 (not 1:200), this gives the doubled outcome of the experiment with the same possible range of concentrations as with the dsDNA HS Assay Kit Q32854 kit by Invitrogen. This procedure is described step by step below:
Step 1. Prepare the 1x TE buffer by diluting the 20x TE concentrate 1:20 with deionized water. Prepare the Pico488 working solution by diluting the Pico488 DNA quantification solution 1:400 (not 1:200) with 1x TE buffer (you need 200 μL Pico488 working solution for each standard and sample). Prepare the DNA standard #2 by diluting the DNA quantification standard (100 ng/mkl) 1:10 in 1x TE buffer.
Step 2. Set up two 0.5 ml tubes (thin-walled and optical-transparent tube) for the standards and one tube for each sample.
Step 3. Prepare standards and samples for concentration measurement. For the preparation of standard #1, mix 190 mkl of Pico488 working solution and 10 mkl of 1x TE buffer in 0.5 ml tube. For the preparation of standard #2, mix 190 mkl of Pico488 working solution and 10 mkl of Standard 2 (from Step 1) in a 0.5 ml tube. For the preparation of samples, mix 180-199 mkl of Pico488 working solution and 1-20 mkl of user DNA sample (the total volume should be 200 μl) in a 0.5 ml tube. Vortex all tubes for 2-3 seconds and incubate the tubes for 3-5 minutes at room temperature.
The next steps should be carried out according to the instructions to the fluorimeter. Depending on the version of the fluorimeter, the names of menu items may differ from the ones below (specified for fluorometer 1).
Step 4. On the Home screen of the fluorometer, you should choose Quant-It dsDNA HS (DNA High Sensitivity) as the assay type. Press "Go"
Step 5. With each preparation of the Pico488 working solution, you must calibrate the fluorimeter. Select "Run new calibration" and press "Go".
Step 6. Insert the tube containing Standard #1 into the sample chamber, close the lid, then press "Go". When the reading is complete (~3 seconds), remove Standard #1. Insert the tube containing Standard #2 into the sample chamber, close the lid, then press"Go". When the reading is complete, remove Standard #2.
Step 7. Insert the tube containing the User sample into the sample chamber, close the lid, then press "Go". On the screen, you will see the QF value. Calculate the concentration of the sample by the formula: Concentration of the sample = QF value x 200/sample volume.