Restriction Enzymes for Droplet Digital PCR (ddPCR)

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


 
Restriction Enzyme Recognition Site
Acc65I G/GTACC
AclI AA/GCTT
AflII C/TTAAG
AluI* AG/CT
ApaI GGGCC/C
AvrII C/CTAGG
BamHI-HF G/GATCC
BglII A/GATCT
BsoBI C/YCGRG
BssSI C/ACGAG
CviAII C/ATG
CviQI* G/TAC
DpnII /GATC
EaeI Y/GGCCR
EcoRI HF G/AATTC
FatI /CATG
HaeIII* GG/CC
HindIII HF* A/AGCTT
HinfI G/ANTC
HpaI GTT/AAC
HphI GGTGA(N)8/
Hpy188I TCN/GA
Hpy188III TC/NNGA
HpyCH4V TG/CA
KpnI-HF GGTAC/C
MfeI HF C/AATTG
MscI TGG/CCA
MseI* T/TAA
NciI CC/SGG
NcoI HF C/CATGG
NlaIII CATG/
RsaI GT/AC
SacI HF GAGCT/C
SphI HF GCATG/C
TfiI G/AWTC
XhoI C/TCGAG

 

*Recommended by Bio-Rad®

HF® is a registered trademark of New England Biolabs, Inc.
TIME-SAVER™ is a trademark of New England Biolabs, Inc.
BIO-RAD® is a registered trademark of Bio-Rad Laboratories, Inc.