Restriction Enzymes for Droplet Digital PCR (ddPCR)
New England Biolabs (NEB) has evaluated a number of restriction enzymes for simple genomic DNA fragmentation in droplet digital PCR assays. This list is not meant to be exhaustive, but provides a starting point for validated enzymes with some simple usage guidelines. Each restriction enzyme listed below was successfully used for a ddPCR digest directly in the Bio-Rad® Droplet Digital SuperMix. The reaction was set up at room temperature with 200 ng human genomic DNA, and no additional incubation step or pre-emulsion inactivation.
Tips for Restriction Enzyme Selection
Avoid enzymes blocked by CpG methylation
Do not use an enzyme with a recognition site present in the target amplicon
While fragment size does not impose strong limitations on enzyme choice, it is best to avoid targets containing fragments >50 kb
If available, we recommend choosing the High Fidelity (HF®) version of an enzyme
For digestion directly in the ddPCR reaction, we recommend choosing a Time-Saver™ Qualified enzyme
Restriction Enzymes in Droplet Digital PCR
Droplet digital PCR is a method for accurately quantitating copies of DNA or RNA in a sample. Each PCR reaction is separated into thousands or millions of droplets for analysis.