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

NEBTV Episode 15: 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 hundreds or thousands droplet for analysis. Restriction enzymes can be used to digest DNA into manageable sizes and increase accuracy. Learn more at <a href="http://www.neb.com/DropletDigitalPCR">www.neb.com/DropletDigitalPCR</a>.

  1. Digestion is recommended whenever DNA input is greater than 75 ng 
  2. Setup 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