For your convenience, New England Biolabs offers a simple 4 buffer system. A color-coded 10X NEBuffer is supplied with every restriction endonuclease, ensuring 100% activity. Over 170 restriction enzymes exhibit 100% activity in NEBuffer 4, resulting in increased efficiency, flexibility and ease of use especially when performing double digests.
To help select the best conditions for double digests, this chart shows the optimal (supplied) NEBuffer and approximate activity in the four standard NEBuffers and NEBuffer EcoRI for each enzyme. If BSA is supplied with an enzyme, it is included in all NEBuffer activity reactions. In addition, the table shows recommended reaction temperature, heat inactivation temperature, recommended diluent buffer and whether the enzyme is Time-Saver qualified (i.e., cleaves substrate in 5 minutes under recommended conditions). Note: The values listed in this table are approximate. They were obtained using each enzyme's specific unit assay substrate DNA.
Please check out other technical reference information related to restriction enzymes:
Double Digestion | Heat Inactivation | Activity at 37°C | Diluent Buffers | Time-Saver Enzymes | High Fidelity (HF) Restriction Enzymes
Click for the Chart Legend, Icon Description
| Enzyme | Supplied NEBuffer | % Activity in NEBuffers | HeatInac. degC | Incu.Temp. | Dilu-ent | |||||
| 1 | 2 | 3 | 4 | EcoRI | ||||||
| AatII | | NEBuffer 4 | 0 | 50 | 50 | 100 | 25 | 65 | 37 | A |
| Acc65I | | NEBuffer 3 + BSA | 10 | 75 | 100 | 25 | 50 | 65 | 37 | A |
| AccI | | NEBuffer 4 | 50 | 50 | 10 | 100 | 5 | 80 | 37 | A |
| AciI | | NEBuffer 3 | 25 | 50 | 100 | 50 | 100 | 65 | 37 | A |
| AclI | | NEBuffer 4 + BSA | 10 | 10 | 0 | 100 | 5 | No | 37 | B |
| AcuI | | NEBuffer 4 + SAM | 50 | 100 | 50 | 100 | 100 | 65 | 37 | B |
| AfeI | | NEBuffer 4 | 25 | 50 | 25 | 100 | 100 | 65 | 37 | B |
| AflII | | NEBuffer 4 + BSA | 50 | 100 | 25 | 100 | 0 | 65 | 37 | A |
| AflIII | | NEBuffer 3 + BSA | 25 | 75 | 100 | 50 | 100 | 80 | 37 | B |
| AgeI | | NEBuffer 1 | 100 | 50 | 10 | 75 | 50 | 65 | 37 | C |
| AgeI-HF&trade | | NEBuffer 4 + BSA | 100 | 50 | 10 | 100 | 25 | 65 | 37 | A |
| AhdI | | NEBuffer 4 + BSA | 25 | 75 | 0 | 100 | 10 | 65 | 37 | A |
| AleI | | NEBuffer 4 | 10 | 25 | 10 | 100 | 0 | 65 | 37 | A |
| AluI | | NEBuffer 4 | 100 | 100 | 75 | 100 | 100 | 65 | 37 | B |
| AlwI | | NEBuffer 4 | 50 | 100 | 10 | 100 | 5 | 65 | 37 | A |
| AlwNI | | NEBuffer 4 | 10 | 100 | 100 | 100 | 10 | 65 | 37 | A |
| ApaI | | NEBuffer 4 + BSA | 25 | 50 | 0 | 100 | 5 | 65 | 25 | A |
| ApaLI | | NEBuffer 4 + BSA | 100 | 100 | 10 | 100 | 5 | No | 37 | A |
| ApeKI | | NEBuffer 3 | 25 | 75 | 100 | 50 | 100 | No | 75 | B |
| ApoI | | NEBuffer 3 + BSA | 10 | 75 | 100 | 75 | 100 | 80 | 50 | A |
| AscI | | NEBuffer 4 | 0 | 10 | 10 | 100 | 5 | 65 | 37 | A |
| AseI | | NEBuffer 3 | NR | 75 | 100 dd | NR | 50 | 65 | 37 | B |
| AsiSI | | NEBuffer 4 + BSA | 50 | 100 | 100 | 100 | 0 | 80 | 37 | B |
| AvaI | | NEBuffer 4 | 10 | 75 | 10 | 100 | 50 | 80 | 37 | A |
| AvaII | | NEBuffer 4 | 50 | 75 | 10 | 100 | 50 | 65 | 37 | A |
| AvrII | | NEBuffer 4 | 100 | 100 | 50 | 100 | 50 | 80 | 37 | B |
| Enzyme top | Supplied NEBuffer | B1 | B2 | B3 | B4 | EcoRI | Heat Inac. | Incu.Temp. | Dilu-ent | |
| BaeGI | | NEBuffer 1 | 100 | 75 | 10 | 50 | 100 | 65 | 37 | A |
| BaeI | NEBuffer 4 + BSA + SAM | 50 | 100 | 50 | 100 | 40 | 65 | 25 | A | |
| BamHI | | NEBuffer 3 + BSA | 75 | 100 | 100 dd | 100 | 100 | No | 37 | A |
| BamHI-HF&trade | | NEBuffer 4 | 100 | 50 | 10 | 100 | 25 | No | 37 | A |
| BanI | | NEBuffer 4 + BSA | 50 | 100 | 50 | 100 | 25 | 65 | 37 | A |
| BanII | | NEBuffer 4 | 100 | 10 | 50 | 100 | 100 | 80 | 37 | A |
| BbsI | NEBuffer 2 | 100 | 100 | 25 | 75 | 50 | 65 | 37 | B | |
| BbvCI | | NEBuffer 4 | 50 | 100 | 10 | 100 | 100 | 80 | 37 | A |
| BbvI | | NEBuffer 2 | 100 | 100 | 25 | 75 | 100 | 65 | 37 | B |
| BccI | | NEBuffer 1 + BSA | 100 | 50 | 10 | 50 | 15 | 65 | 37 | A |
| BceAI | | NEBuffer 3 + BSA | 100 | 100 | 100 | 100 | 100 | 65 | 37 | A |
| BcgI | | NEBuffer 3 + SAM | NR | NR | 100 dd | NR | 50 | 65 | 37 | A |
| BciVI | | NEBuffer 4 | 100 | 50 | 0 | 100 | 25 | 65 | 37 | C |
| BclI | | NEBuffer 3 | 50 | 100 | 100 dd | 75 | 100 | No | 50 | A |
| BcoDI | | NEBuffer 4 | 50 | 75 | 75 | 100 | 100 | 65 | 37 | B |
| BfaI | NEBuffer 4 | 75 | 50 | 10 | 100 | 0 | 80 | 37 | B | |
| BfuAI | | NEBuffer 3 | 0 | 75 | 100 | 10 | 100 | 65 | 50 | B |
| BfuCI | NEBuffer 4 + BSA | 100 | 50 | 25 | 100 | 15 | 80 | 37 | B | |
| BglI | | NEBuffer 3 | 50 | 75 | 100 | 50 | 100 | 65 | 37 | B |
| BglII | | NEBuffer 3 | 10 | 75 | 100 | 10 | 100 | No | 37 | A |
| BlpI | | NEBuffer 4 | 50 | 100 | 10 | 100 | 50 | No | 37 | A |
| BmgBI | | NEBuffer 3 + BSA | 0 | 25 | 100 | 10 | 50 | 65 | 37 | C |
| BmrI | | NEBuffer 2 | 75 | 100 | 75 | 100 | 50 | 65 | 37 | B |
| BmtI | | NEBuffer 2 | 25 | 100 | 25 | 50 | 100 | 65 | 37 | B |
| BpmI | | NEBuffer 3 + BSA | 75 | 100 | 100 | 100 | 100 | 65 | 37 | B |
| Bpu10I | | NEBuffer 3 | 10 | 25 | 100 | 25 | 100 | 80 | 37 | B |
| BpuEI | | NEBuffer 4 + SAM | 50 | 100 | 10 | 100 | 100 | 65 | 37 | B |
| BsaAI | | NEBuffer 4 | 100 | 100 | 100 | 100 | 100 | No | 37 | C |
| BsaBI | | NEBuffer 4 | 50 | 100 | 75 | 100 | 100 | 80 | 60 | B |
| BsaHI | | NEBuffer 4 + BSA | 50 | 100 | 100 | 100 | 100 | 80 | 37 | A |
| BsaI | | NEBuffer 4 + BSA | 75 | 75 | 100 | 100 | 100 | 65 | 37 | B |
| BsaI-HF&trade | | NEBuffer 4 + BSA | 25 | 25 | 10 | 100 | 10 | 65 | 37 | B |
| BsaJI | | NEBuffer 4 | 100 | 100 | 100 | 100 | 100 | 80 | 60 | A |
| BsaWI | | NEBuffer 4 + BSA | 50 | 100 | 50 | 100 | 40 | 80 | 60 | A |
| BsaXI | | NEBuffer 4 | 75 | 100 | 10 | 100 | 15 | No | 37 | B |
| BseRI | | NEBuffer 4 | 100 | 100 | 75 | 100 | 100 | 65 | 37 | A |
| BseYI | | NEBuffer 3 | 10 | 50 | 100 | 50 | 100 | 65 | 37 | B |
| BsgI | | NEBuffer 4 + SAM | 50 | 75 | 50 | 100 | 20 | 65 | 37 | B |
| BsiEI | | NEBuffer 4 + BSA | 50 | 100 | 10 | 100 | 5 | 80 | 60 | A |
| BsiHKAI | NEBuffer 4 + BSA | 50 | 100 | 100 | 100 | 25 | No | 65 | B | |
| BsiWI | | NEBuffer 3 | 100 | 100 | 100 | 25 | 100 | 80 | 55 | A |
| BslI | | NEBuffer 3 | 10 | 50 | 100 | 75 | 100 | 80 | 55 | A |
| BsmAI | | NEBuffer 4 | 50 | 100 | 100 | 100 | 100 | 80 | 55 | B |
| BsmBI | | NEBuffer 3 | 75 | 100 | 100 | 100 | 100 | 80 | 55 | B |
| BsmFI | | NEBuffer 4 + BSA | 25 | 50 | 50 | 100 | 15 | 80 | 65 | A |
| BsmI | | NEBuffer 4 | 75 | 100 | 75 | 100 | 5 | 80 | 65 | A |
| BsoBI | | NEBuffer 2 | 10 | 100 | 100 | 50 | 100 | No | 37 | A |
| Bsp1286I | | NEBuffer 4 + BSA | 25 | 25 | 25 | 100 | 10 | 65 | 37 | A |
| BspCNI | NEBuffer 4 + BSA + SAM | 100 | 75 | 10 | 100 | 15 | 80 | 25 | A | |
| BspDI | | NEBuffer 4 | 25 | 75 | 50 | 100 | 100 | 65 | 37 | A |
| BspEI | | NEBuffer 3 | 0 | 10 | 100 | 0 | 100 | 80 | 37 | B |
| BspHI | | NEBuffer 4 | 10 | 100 | 50 | 100 | 5 | 65 | 37 | A |
| BspMI | | NEBuffer 3 | NR | NR | 100 | NR | 100 | 65 | 37 | B |
| BspQI | | NEBuffer 4 | 10 | 50 | 100 | 100 | 100 | 80 | 50 | B |
| BsrBI | | NEBuffer 4 | 50 | 100 | 100 | 100 | 100 | 80 | 37 | A |
| BsrDI | | NEBuffer 2 + BSA | 50 | 100 | 50 | 75 | 50 | 80 | 65 | A |
| BsrFI | | NEBuffer 4 | 10 | 100 | 100 | 100 | 100 | No | 37 | C |
| BsrGI | | NEBuffer 2 + BSA | 25 | 100 | 10 | 100 | 50 | 80 | 37 | A |
| BsrI | | NEBuffer 3 | 0 | 50 | 100 | 10 | 100 | 80 | 65 | B |
| BssHII | | NEBuffer 3 | 100 | 100 | 100 dd | 100 | 100 | 80 | 50 | B |
| BssKI | | NEBuffer 3 + BSA | 0 | 50 | 100 | 50 | 100 | 80 | 60 | A |
| BssSI | | NEBuffer 3 + BSA | 0 | 50 | 100 dd | 10 | 100 | 80 | 37 | B |
| BstAPI | | NEBuffer 4 + BSA | 25 | 100 | 100 | 100 | 100 | 80 | 60 | A |
| BstBI | | NEBuffer 4 | 75 | 50 | 25 | 100 | 50 | No | 65 | A |
| BstEII | | NEBuffer 3 + BSA | 50 | 75 | 100 | 75 | 100 | No | 60 | A |
| BstNI | | NEBuffer 2 + BSA | 10 | 100 | 100 | 75 | 50 | No | 60 | A |
| BstUI | | NEBuffer 4 | 100 | 100 | 50 | 100 | 100 | No | 60 | A |
| BstXI | | NEBuffer 3 | 0 | 50 | 100 | 25 | 100 | 65 | 37 | B |
| BstYI | | NEBuffer 2 | 50 | 100 | 75 | 100 | 50 | 80 | 60 | A |
| BstZ17I | | NEBuffer 4 | NR | NR | 100 | 100 | 100 | No | 37 | B |
| Bsu36I | | NEBuffer 3 + BSA | 0 | 25 | 100 | 0 | 15 | 80 | 37 | A |
| BtgI | | NEBuffer 3 + BSA | 25 | 50 | 100 | 100 | 100 | 80 | 37 | B |
| BtgZI | | NEBuffer 4 + BSA | 10 | 25 | 0 | 100 | 50 | 80 | 60 | A |
| BtsCI | | NEBuffer 4 | 50 | 100 | 50 | 100 | 25 | No | 50 | B |
| BtsI | | NEBuffer 4 + BSA | 100 | 50 | 50 | 100 | 25 | 80 | 55 | A |
| BtsIMutI | | NEBuffer 4 + BSA | 100 | 50 | 10 | 100 | - | 80 | 55 | A |
| Enzyme top | Supplied NEBuffer | B1 | B2 | B3 | B4 | EcoRI | Heat Inac. | Incu.Temp. | Dilu-ent | |
| Cac8I | NEBuffer 4 | 50 | 75 | 100 | 100 | 50 | 65 | 37 | B | |
| ClaI | | NEBuffer 4 + BSA | 10 | 50 | 50 | 100 | 50 | 65 | 37 | A |
| CspCI | | NEBuffer 4 + SAM | 10 | 100 | 10 | 100 | 20 | 65 | 37 | A |
| CviAII | | NEBuffer 4 + BSA | 75 | 25 | 10 | 100 | 25 | 65 | 25 | C |
| CviKI-1 | | NEBuffer 4 | 10 | 50 | 25 | 100 | 100 | 80 | 37 | A |
| CviQI | | NEBuffer 3 + BSA | 75 | 100 | 100 | 75 | 0 | No | 25 | B |
| Enzyme top | Supplied NEBuffer | B1 | B2 | B3 | B4 | EcoRI | Heat Inac. | Incu.Temp. | Dilu-ent | |
| DdeI | | NEBuffer 3 | 75 | 100 | 100 | 75 | 100 | 65 | 37 | A |
| DpnI | | NEBuffer 4 | 100 | 100 | 75 | 100 | 100 | 80 | 37 | B |
| DpnII | | NEBuffer DpnII | NR | NR | 100 | NR | 100 | 65 | 37 | B |
| DraI | | NEBuffer 4 | 75 | 75 | 50 | 100 | 100 | 65 | 37 | A |
| DraIII | | NEBuffer 3 + BSA | 100 | 75 | 100 | 25 | 50 | 65 | 37 | B |
| DraIII-HF&trade | | NEBuffer 4 | 0 | 50 | 10 | 100 | 50 | No | 37 | B |
| DrdI | NEBuffer 4 + BSA | 25 | 50 | 10 | 100 | 100 | 65 | 37 | A | |
| Enzyme top | Supplied NEBuffer | B1 | B2 | B3 | B4 | EcoRI | Heat Inac. | Incu.Temp. | Dilu-ent | |
| EaeI | | NEBuffer 4 | 100 | 100 | 50 | 100 | 100 | 80 | 37 | A |
| EagI | | NEBuffer 3 | 10 | 25 | 100 | 10 | 100 | 65 | 37 | C |
| EagI-HF&trade | | NEBuffer 4 | 25 | 100 | 100 | 100 | 100 | 65 | 37 | B |
| EarI | | NEBuffer 4 | 100 | 100 | 50 | 100 | 100 | 65 | 37 | A |
| EciI | NEBuffer 2 + BSA | 50 | 100 | 100 | 50 | 100 | 65 | 37 | A | |
| Eco53kI | | NEBuffer 4 | 100 | 50 | 25 | 100 | 10 | 65 | 37 | A |
| EcoNI | | NEBuffer 4 | 100 | 100 | 75 | 100 | 100 | 65 | 37 | A |
| EcoO109I | | NEBuffer 4 + BSA | 100 | 100 | 75 | 100 | 15 | 65 | 37 | A |
| EcoP15I | | NEBuffer 3 + BSA + ATP | 75 | 100 | 100 | 100 | 50 | 65 | 37 | A |
| EcoRI | | NEBuffer EcoRI | 100 | 100 | 100 | 100 | 100 | 65 | 37 | C |
| EcoRI-HF&trade | | NEBuffer 4 | 10 | 100 | 0 | 100 | 5 | 65 | 37 | C |
| EcoRV | | NEBuffer 3 + BSA | 50 | 75 | 100 | 50 | 50 | 80 | 37 | A |
| EcoRV-HF&trade | | NEBuffer 4 | 25 | 100 | 100 | 100 | 100 | 65 | 37 | B |
| Enzyme top | Supplied NEBuffer | B1 | B2 | B3 | B4 | EcoRI | Heat Inac. | Incu.Temp. | Dilu-ent | |
| FatI | | NEBuffer 2 | 10 | 100 | 50 | 50 | 100 | 65 | 55 | A |
| FauI | | NEBuffer 4 | 100 | 50 | 10 | 100 | 50 | 65 | 55 | A |
| Fnu4HI | | NEBuffer 4 | 10 | 25 | 25 | 100 | 10 | 65 | 37 | A |
| FokI | | NEBuffer 4 | 100 | 100 | 75 | 100 | 25 | 65 | 37 | A |
| FseI | | NEBuffer 4 + BSA | 100 | 75 | 0 | 100 | 50 | 65 | 37 | B |
| FspEI | | NEBuffer 4 + BSA | NR | NR | NR | 100 | - | 65 | 37 | B |
| FspI | | NEBuffer 4 | 10 | 75 | 10 | 100 | 50 | 65 | 37 | C |
| Enzyme top | Supplied NEBuffer | B1 | B2 | B3 | B4 | EcoRI | Heat Inac. | Incu.Temp. | Dilu-ent | |
| HaeII | | NEBuffer 4 + BSA | 75 | 100 | 50 | 100 | 25 | 80 | 37 | A |
| HaeIII | | NEBuffer 4 | 50 | 100 | 25 | 100 | 100 | 80 | 37 | A |
| HgaI | | NEBuffer 1 | 100 | 75 | 50 | 100 | 25 | 65 | 37 | A |
| HhaI | | NEBuffer 4 + BSA | 75 | 100 | 100 | 100 | 100 | 65 | 37 | A |
| HincII | | NEBuffer 3 + BSA | 75 | 100 | 100 | 100 | 100 | 65 | 37 | B |
| HindIII | | NEBuffer 2 | 50 | 100 | 10 | 50 | 25 | 65 | 37 | B |
| HindIII-HF&trade | | NEBuffer 4 | 100 | 10 | 10 | 100 | 10 | 80 | 37 | B |
| HinfI | | NEBuffer 4 | 75 | 100 | 75 | 100 | 100 | 80 | 37 | A |
| HinP1I | | NEBuffer 4 | 100 | 100 | 100 | 100 | 100 | 65 | 37 | A |
| HpaI | | NEBuffer 4 | 25 | 25 | 10 | 100 | 100 | No | 37 | A |
| HpaII | | NEBuffer 1 | 100 | 50 | 10 | 50 | 15 | 65 | 37 | A |
| HphI | | NEBuffer 4 | NR | 75 | 0 | 100 | 10 | 65 | 37 | B |
| Hpy166II | | NEBuffer 4 | 100 | 100 | 50 | 100 | 20 | 65 | 37 | C |
| Hpy188I | | NEBuffer 4 | 50 | 25 | 10 | 100 | 50 | 65 | 37 | A |
| Hpy188III | | NEBuffer 4 + BSA | 100 | 100 | 10 | 100 | 5 | 65 | 37 | A |
| Hpy99I | | NEBuffer 4 + BSA | 10 | 10 | 0 | 100 | 25 | 65 | 37 | A |
| HpyAV | | NEBuffer 4 + BSA | 100 | 100 | 25 | 100 | 50 | 65 | 37 | B |
| HpyCH4III | | NEBuffer 4 | 100 | 50 | 25 | 100 | 20 | 80 | 37 | A |
| HpyCH4IV | | NEBuffer 1 | 100 | 25 | 10 | 25 | 15 | 65 | 37 | A |
| HpyCH4V | | NEBuffer 4 | 50 | 50 | 25 | 100 | 50 | 65 | 37 | B |
| Enzyme top | Supplied NEBuffer | B1 | B2 | B3 | B4 | EcoRI | Heat Inac. | Incu.Temp. | Dilu-ent | |
| I-CeuI | | NEBuffer 4 + BSA | 10 | 10 | 0 | 100 | - | 65 | 37 | B |
| I-SceI | | NEBuffer I-SceI + BSA | 10 | 50 | 50 | 50 | - | 65 | 37 | B |
| Enzyme top | Supplied NEBuffer | B1 | B2 | B3 | B4 | EcoRI | Heat Inac. | Incu.Temp. | Dilu-ent | |
| KasI | | NEBuffer 4 + BSA | 25 | 100 | 0 | 100 | 100 | 65 | 37 | B |
| KpnI | | NEBuffer 1 + BSA | 100 | 75 | 0 | 50 | 25 | No | 37 | A |
| KpnI-HF&trade | | NEBuffer 4 | 100 | 25 | 0 | 100 | 0 | No | 37 | A |
| Enzyme top | Supplied NEBuffer | B1 | B2 | B3 | B4 | EcoRI | Heat Inac. | Incu.Temp. | Dilu-ent | |
| LpnPI | | NEBuffer 4 + BSA | NR | NR | NR | 100 | - | 65 | 37 | B |
| Enzyme top | Supplied NEBuffer | B1 | B2 | B3 | B4 | EcoRI | Heat Inac. | Incu.Temp. | Dilu-ent | |
| MboI | | NEBuffer 4 | 75 | 100 | 100 | 100 | 100 | 65 | 37 | A |
| MboII | | NEBuffer 4 | 100 | 100 | 50 | 100 | 100 | 65 | 37 | C |
| MfeI | | NEBuffer 4 | 75 | 50 | 10 | 100 | 5 | 65 | 37 | A |
| MfeI-HF&trade | | NEBuffer 4 | 75 | 50 | 10 | 100 | 0 | 65 | 37 | A |
| MluCI | | NEBuffer 4 | 100 | 10 | 10 | 100 | 0 | 80 | 37 | A |
| MluI | | NEBuffer 3 | 25 | 75 | 100 | 50 | 100 | 65 | 37 | A |
| MlyI | | NEBuffer 4 + BSA | 50 | 50 | 25 | 100 | 25 | 65 | 37 | A |
| MmeI | | NEBuffer 4 + SAM | NR | NR | NR | 100 dd | 50 | 80 | 37 | B |
| MnlI | | NEBuffer 4 + BSA | 75 | 100 | 50 | 100 | 50 | 65 | 37 | B |
| MscI | | NEBuffer 4 | 75 | 75 | 75 | 100 | 100 | 65 | 37 | B |
| MseI | | NEBuffer 4 + BSA | 75 | 100 | 75 | 100 | 50 | 65 | 37 | A |
| MslI | | NEBuffer 4 | 100 | 100 | 25 | 100 | 10 | 65 | 37 | A |
| MspA1I | | NEBuffer 4 + BSA | 50 | 100 | 50 | 100 | 50 | 65 | 37 | B |
| MspI | | NEBuffer 4 | 75 | 100 | 50 | 100 | 50 | No | 37 | A |
| MspJI | | NEBuffer 4 + BSA | NR | NR | NR | 100 | - | 65 | 37 | B |
| MwoI | | NEBuffer 3 | 10 | 75 | 100 | 75 | 100 | No | 60 | B |
| Enzyme top | Supplied NEBuffer | B1 | B2 | B3 | B4 | EcoRI | Heat Inac. | Incu.Temp. | Dilu-ent | |
| NaeI | | NEBuffer 4 | 100 | 75 | 10 | 100 | 25 | 65 | 37 | A |
| NarI | | NEBuffer 4 | 100 | 75 | 75 | 100 | 50 | 65 | 37 | A |
| Nb.BbvCI | | NEBuffer 2 | 50 | 100 | 10 | 100 | - | 80 | 37 | A |
| Nb.BsmI | | NEBuffer 3 | 25 | 100 | 100 | 25 | - | 80 | 65 | A |
| Nb.BsrDI | | NEBuffer 2 | 10 | 100 | 25 | 50 | - | 80 | 65 | A |
| Nb.BtsI | | NEBuffer 4 + BSA | 75 | 100 | 75 | 100 | - | 80 | 37 | A |
| NciI | | NEBuffer 4 | 100 | 25 | 10 | 100 | 25 | No | 37 | A |
| NcoI | | NEBuffer 3 | 100 | 100 | 100 | 100 | 100 | 65 | 37 | A |
| NcoI-HF&trade | | NEBuffer 4 | 50 | 100 | 10 | 100 | 100 | 80 | 37 | B |
| NdeI | | NEBuffer 4 | 75 | 100 | 75 | 100 | 100 | 65 | 37 | A |
| NgoMIV | | NEBuffer 4 | 100 | 50 | 10 | 100 | 0 | 80 | 37 | A |
| NheI | | NEBuffer 2 + BSA | 100 | 100 | 10 | 100 | 15 | 65 | 37 | C |
| NheI-HF&trade | | NEBuffer 4 + BSA | 100 | 10 | 0 | 100 | 0 | 80 | 37 | C |
| NlaIII | | NEBuffer 4 + BSA | 25 | 25 | 25 | 100 | 5 | 65 | 37 | B |
| NlaIV | | NEBuffer 4 + BSA | 10 | 10 | 10 | 100 | 100 | 65 | 37 | B |
| NmeAIII | | NEBuffer 4 + SAM | 10 | 10 | 0 | 100 | 0 | 80 | 37 | B |
| NotI | | NEBuffer 3 + BSA | 0 | 50 | 100 | 25 | 100 | 65 | 37 | C |
| NotI-HF&trade | | NEBuffer 4 + BSA | 25 | 100 | 25 | 100 | 50 | 65 | 37 | A |
| NruI | | NEBuffer 3 | 0 | 10 | 100 | 10 | 100 | 65 | 37 | A |
| NsiI | | NEBuffer 3 | 10 | 75 | 100 | 25 | 100 | 80 | 37 | A |
| NspI | | NEBuffer 2 + BSA | 100 | 100 | 0 | 100 | 5 | 65 | 37 | A |
| Nt.AlwI | | NEBuffer 2 | 25 | 100 | 25 | 50 | - | 80 | 37 | A |
| Nt.BbvCI | | NEBuffer 4 | 50 | 100 | 10 | 100 | - | 80 | 37 | A |
| Nt.BsmAI | | NEBuffer 4 | 100 | 50 | 10 | 100 | - | 65 | 37 | A |
| Nt.BspQI | | NEBuffer 3 | 10 | 75 | 100 | 25 | - | 80 | 50 | B |
| Nt.BstNBI | | NEBuffer 3 | 0 | 10 | 100 | 10 | - | 80 | 55 | A |
| Nt.CviPII | | NEBuffer 4 | 25 | 100 | 50 | 100 | - | 65 | 37 | A |
| Enzyme top | Supplied NEBuffer | B1 | B2 | B3 | B4 | EcoRI | Heat Inac. | Incu.Temp. | Dilu-ent | |
| PacI | | NEBuffer 1 + BSA | 100 | 75 | 10 | 100 | 5 | 65 | 37 | A |
| PaeR7I | | NEBuffer 4 | 25 | 100 | 10 | 100 | 50 | No | 37 | A |
| PciI | | NEBuffer 3 + BSA | 50 | 75 | 100 | 50 | 0 | 80 | 37 | B |
| PflFI | | NEBuffer 4 + BSA | 0 | 25 | 25 | 100 | 0 | 65 | 37 | A |
| PflMI | | NEBuffer 3 + BSA | 0 | 100 | 100 | 50 | 100 | 65 | 37 | A |
| PhoI | | NEBuffer 3 | 50 | 50 | 100 | 75 | 100 | No | 75 | A |
| PI-PspI | | NEBuffer PI-PspI + BSA | 0 | 10 | 10 | 10 | - | No | 65 | B |
| PI-SceI | | NEBuffer PI-SceI + BSA | 0 | 0 | 0 | 0 | - | 65 | 37 | B |
| PleI | | NEBuffer 4 | 10 | 100 | 100 | 100 | 50 | 65 | 37 | A |
| PmeI | | NEBuffer 4 + BSA | 0 | 50 | 10 | 100 | 5 | 65 | 37 | A |
| PmlI | | NEBuffer 1 + BSA | 100 | 75 | 0 | 75 | 15 | 65 | 37 | A |
| PpuMI | | NEBuffer 4 | 0 | 25 | 0 | 100 | 10 | No | 37 | B |
| PshAI | | NEBuffer 4 + BSA | 50 | 50 | 0 | 100 | 5 | 65 | 37 | A |
| PsiI | | NEBuffer 4 | 10 | 100 | 10 | 100 | 10 | 65 | 37 | B |
| PspGI | | NEBuffer 4 | 75 | 100 | 50 | 100 | 100 | No | 75 | A |
| PspOMI | | NEBuffer 4 | 25 | 25 | 10 | 100 | 15 | 65 | 37 | B |
| PspXI | | NEBuffer 4 | 0 | 100 | 10 | 100 | 50 | 80 | 37 | B |
| PstI | | NEBuffer 3 + BSA | 75 | 75 | 100 | 50 | 50 | 80 | 37 | C |
| PstI-HF&trade | | NEBuffer 4 | 10 | 75 | 50 | 100 | 25 | 80 | 37 | C |
| PvuI | | NEBuffer 3 + BSA | 10 | 75 | 100 | 10 | 100 | 80 | 37 | B |
| PvuI-HF&trade | | NEBuffer 4 + BSA | 25 | 100 | 100 | 100 | 100 | No | 37 | B |
| PvuII | | NEBuffer 2 | 100 | 100 | 100 | 100 | 100 | No | 37 | B |
| PvuII-HF&trade | | NEBuffer 4 | 0 | 25 | 0 | 100 | 0 | 80 | 37 | B |
| Enzyme top | Supplied NEBuffer | B1 | B2 | B3 | B4 | EcoRI | Heat Inac. | Incu.Temp. | Dilu-ent | |
| RsaI | | NEBuffer 4 | 100 | 100 | 50 | 100 | 15 | 65 | 37 | A |
| RsrII | | NEBuffer 4 | 25 | 75 | 10 | 100 | 25 | 65 | 37 | C |
| Enzyme top | Supplied NEBuffer | B1 | B2 | B3 | B4 | EcoRI | Heat Inac. | Incu.Temp. | Dilu-ent | |
| SacI | | NEBuffer 1 + BSA | 100 | 50 | 10 | 100 | 15 | 65 | 37 | A |
| SacI-HF&trade | | NEBuffer 4 + BSA | 100 | 50 | 10 | 100 | 15 | 65 | 37 | A |
| SacII | | NEBuffer 4 | 25 | 75 | 10 | 100 | 100 | 65 | 37 | A |
| SalI | | NEBuffer 3 + BSA | 0 | 0 | 100 | 0 | 50 | 65 | 37 | A |
| SalI-HF&trade | | NEBuffer 4 | 10 | 100 | 100 | 100 | 100 | 65 | 37 | A |
| SapI | | NEBuffer 4 | 75 | 50 | 0 | 100 | 50 | 65 | 37 | C |
| Sau3AI | | NEBuffer 1 + BSA | 100 | 50 | 10 | 100 | 5 | 65 | 37 | A |
| Sau96I | | NEBuffer 4 | 50 | 100 | 100 | 100 | 50 | 80 | 37 | A |
| SbfI | | NEBuffer 4 | 75 | 50 | 0 | 100 | 25 | 65 | 37 | A |
| SbfI-HF&trade | | NEBuffer 4 | 50 | 25 | 0 | 100 | 0 | 65 | 37 | B |
| ScaI | | NEBuffer 3 | NR | NR | 100 dd | NR | 50 | 80 | 37 | A |
| ScaI-HF&trade | | NEBuffer 4 | 100 | 100 | 10 | 100 | 15 | 65 | 37 | B |
| ScrFI | | NEBuffer 4 | 100 | 100 | 100 | 100 | 100 | 65 | 37 | C |
| SexAI | | NEBuffer 4 + BSA | 100 | 75 | 50 | 100 | 50 | 65 | 37 | A |
| SfaNI | | NEBuffer 3 | 0 | 75 | 100 | 25 | 50 | 65 | 37 | B |
| SfcI | | NEBuffer 4 + BSA | 75 | 50 | 10 | 100 | 25 | 65 | 37 | B |
| SfiI | | NEBuffer 4 + BSA | 0 | 100 | 10 | 100 | 15 | No | 50 | C |
| SfoI | | NEBuffer 4 | 25 | 100 | 50 | 100 | 100 | No | 37 | B |
| SgrAI | | NEBuffer 4 | 100 | 50 | 10 | 100 | 50 | 65 | 37 | A |
| SmaI | | NEBuffer 4 | 0 | 0 | 0 | 100 | 0 | 65 | 25 | A |
| SmlI | | NEBuffer 4 + BSA | 25 | 75 | 25 | 100 | 50 | No | 55 | A |
| SnaBI | | NEBuffer 4 + BSA | 25 | 50 | 25 | 100 | 5 | 80 | 37 | A |
| SpeI | | NEBuffer 4 + BSA | 75 | 100 | 25 | 100 | 50 | 80 | 37 | C |
| SphI | | NEBuffer 2 | 100 | 100 | 50 | 100 | 50 | 65 | 37 | B |
| SphI-HF&trade | | NEBuffer 4 | 50 | 25 | 10 | 100 | 0 | 65 | 37 | B |
| SspI | | NEBuffer SspI | 50 | 100 | 50 | 50 | 100 | 65 | 37 | C |
| SspI-HF&trade | | NEBuffer 4 | 25 | 100 | 0 | 100 | 25 | 65 | 37 | B |
| StuI | | NEBuffer 4 | 100 | 100 | 50 | 100 | 50 | 65 | 37 | A |
| StyD4I | | NEBuffer 2 | 10 | 100 | 100 | 100 | 20 | 65 | 37 | B |
| StyI | | NEBuffer 3 + BSA | 25 | 75 | 100 | 10 | 15 | 65 | 37 | A |
| StyI-HF&trade | | NEBuffer 4 | 25 | 100 | 25 | 100 | 0 | 65 | 37 | A |
| SwaI | | NEBuffer 3 + BSA | 10 | 10 | 100 | 10 | 15 | 65 | 25 | B |
| Enzyme top | Supplied NEBuffer | B1 | B2 | B3 | B4 | EcoRI | Heat Inac. | Incu.Temp. | Dilu-ent | |
| TaqαI | | NEBuffer 4 + BSA | 50 | 75 | 100 | 100 | 100 | 80 | 65 | B |
| TfiI | | NEBuffer 4 | 100 | 100 | 100 | 100 | 100 | No | 65 | C |
| TliI | | NEBuffer 3 + BSA | 25 | 25 | 100 | 10 | 50 | No | 75 | B |
| TseI | NEBuffer 4 | 75 | 100 | 100 | 100 | 100 | No | 65 | B | |
| Tsp45I | NEBuffer 1 + BSA | 100 | 25 | 0 | 75 | 50 | No | 65 | A | |
| Tsp509I | | NEBuffer 1 | 100 | 100 | 100 | NR | 100 | No | 65 | A |
| TspMI | | NEBuffer 4 | 50 | 75 | 50 | 100 | 20 | No | 75 | B |
| TspRI | | NEBuffer 4 + BSA | 25 | 50 | 25 | 100 | 5 | No | 65 | B |
| Tth111I | | NEBuffer 4 | 50 | 25 | 25 | 100 | 100 | No | 65 | B |
| Enzyme top | Supplied NEBuffer | B1 | B2 | B3 | B4 | EcoRI | Heat Inac. | Incu.Temp. | Dilu-ent | |
| XbaI | | NEBuffer 4 + BSA | 0 | 100 | 75 | 100 | 15 | 65 | 37 | A |
| XcmI | | NEBuffer 2 | 10 | 100 | 50 | 50 | 20 | 65 | 37 | C |
| XhoI | | NEBuffer 4 + BSA | 75 | 100 | 100 | 100 | 100 | 65 | 37 | A |
| XmaI | | NEBuffer 4 + BSA | 25 | 50 | 0 | 100 | 25 | 65 | 37 | A |
| XmnI | | NEBuffer 4 + BSA | 100 | 100 | 50 | 100 | 5 | 65 | 37 | A |
| Enzyme top | Supplied NEBuffer | B1 | B2 | B3 | B4 | EcoRI | Heat Inac. | Incu.Temp. | Dilu-ent | |
| ZraI | | NEBuffer 4 | 100 | 25 | 10 | 100 | 10 | No | 37 | B |
Note:
1. SspI Unique Buffer has the same composition as EcoRI Unique Buffer.
2. The values listed in this table are approximate. They were obtained using each enzyme's specific unit assay substrate DNA