Digital PCR Support—Troubleshooting

First you need to know the amount of nanograms per copy of your sample. In this example, we'll use human genomic DNA as the source of DNA.
Now you need to multiply ng/copy by copies/µL. For our example, we were given 2,500 copies/µL of human gDNA
                  2,500 copies/µL x 0.0033 ng/copy = 8.25 ng/µL

If you enter all of the necessary dilution factors into the software, the software will give you the copies/µL in your stock. There are two dilutions you need to take into account. The first is the dilution of the sample in the reaction. The second is the dilution of the stock that you make before adding it to the digital PCR reaction.

For example, say you want to add 1 µL of a sample that has been diluted 1:10 from the stock.

If you add 1 µL of your sample to a reaction that has a final volume of 16 µL, the dilution factor of the sample is 1/16, or 0.0625. Since the stock has also been diluted 1:10 (0.1), you also need to factor this in. The final dilution factor to enter into the software is 0.0625 * 0.1 = 0.00625 (1:160). As you can see below, you can use either annotation to indicate the dilution factor in the software.

For example, if you enter that value into the “Dilution” column in AnalysisSuite™ Software, the software will give you the copies/µL in your starting material (stock).

AnalysisSuite™ Software is optimized for Google® Chrome™ v4.0 or later. Firefox also works, but Internet Explorer is not recommended.

You can use the column headers to sort your data as ascending/descending or nested sorted. If you need more flexibility in the graphing display, you can export the data as a .csv file so that you can recreate your graphs exactly how you want them.

For digital PCR, it is important to make sure that your samples are in the “digital range” (sufficiently diluted such that some wells will have template and some wells will not). If you run a chip or plate with no sample at all, then you are not in the digital range. This can then cause problems in the analysis. Check that the threshold is being set properly in the AnalysisSuite™ Software. You may need to set it manually.

Use the example calculations in Figure 9 from this Application Note.