Protocol for Direct Digestion of gDNA during droplet digital PCR (ddPCR)

1. Digestion is recommended whenever DNA input is greater than 75 ng
2. Set up reactions at room temperature to allow digestion
3. Prepare reaction mixes as you would for a standard ddPCR reaction. Add 0.5–1 μl of each restriction enzyme (5–20 units, depending on enzyme concentration) to the reaction mixture
4. After setup, simply continue droplet generation as normal
5. Restriction enzyme will be inactivated during first PCR denaturation step

Note: If user would like to digest prior to droplet digital PCR, please complete the following steps:

1. Set up restriction enzyme digests in the recommended NEBuffer™ 
2. Use 10 units of restriction enzyme per microgram of DNA sample
3. Incubate at incubation temperature for 5–60 minutes as recommended for each enzyme 
4. Enzyme can be heat inactivated if desired, but this is not required 
5. No cleanup is necessary after digestion; sample can be directly added to ddPCR reactions 
6. Avoid carrying over more than a 1/10 amount of the restriction digest mixture to the ddPCR reaction