Protocol for Direct Digestion of gDNA for ddPCR
- Digestion is recommended whenever DNA input is greater than 75 ng
- Assemble ddPCR reactions at room temperature, this allows the restriction enzyme to digest the gDNA during the reaction set-up period
- 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
- After set-up, simply continue droplet generation as normal
- Restriction enzyme will be inactivated during first PCR denaturation step
Protocol for Digestion Prior to ddPCR
- Set-up restriction enzyme digests in the recommended NEBuffer
- Use 10 units of restriction enzyme per microgram of DNA sample
- Incubate for 5–60 minutes at the recommended reaction temperature for the respective enzyme
- Enzyme can be heat inactivated if desired, but this is not required
- No cleanup is necessary after digestion; sample can be directly added to ddPCR reactions
- Avoid carrying over more than a 1/10 amount of the restriction digest mixture to the ddPCR reaction
*Recommended by Bio-Rad®
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