liminfoRestriction Enzyme Database
Free reference guide: Restriction Enzyme Database
About Restriction Enzyme Database
The Restriction Enzyme Database is a comprehensive reference for Type II restriction endonucleases used in molecular cloning, DNA manipulation, and genetic engineering. It covers 15+ commonly used 6-cutter enzymes (EcoRI G^AATTC, BamHI G^GATCC, HindIII A^AGCTT, XhoI C^TCGAG, NdeI CA^TATG, KpnI GGTAC^C, SalI, PstI, SpeI, NheI, BglII, EcoRV, SmaI), 8-cutter rare cutters (NotI GC^GGCCGC cutting every ~65,536 bp, PacI, SwaI), and special enzymes.
The reference includes detailed cut site patterns showing 5-prime overhangs, 3-prime overhangs, and blunt ends, along with compatible end pairs (BamHI/BglII, XhoI/SalI, SpeI/NheI). It covers reaction conditions including CutSmart universal buffer composition (50 mM KOAc, 20 mM Tris-OAc, 10 mM Mg(OAc)2), star activity causes and prevention with High-Fidelity enzymes, Dam/Dcm methylation effects on enzyme activity, and T4 DNA Ligase protocols for sticky-end and blunt-end ligation.
Special enzyme sections detail DpnI for methylated DNA digestion in site-directed mutagenesis, Type IIS enzymes (BsaI, BsmBI, BbsI) for Golden Gate and MoClo cloning with custom sticky ends, nicking enzymes for isothermal amplification, homing endonucleases with 14-40 bp recognition sequences, and isoschizomer/neoschizomer pairs. NEB catalog numbers are included for easy ordering.
Key Features
- Complete recognition sequences and cut site diagrams for 15+ Type II 6-cutter restriction enzymes with NEB catalog numbers
- Rare 8-cutter enzyme reference (NotI, PacI, SwaI) with cutting frequency calculations for genome mapping applications
- Compatible cohesive end pairs mapped between enzymes (BamHI/BglII, XhoI/SalI, SpeI/NheI) for flexible cloning strategies
- CutSmart universal buffer composition and compatibility data covering 95%+ of NEB restriction enzymes
- Star activity causes, prevention strategies, and High-Fidelity (HF) enzyme alternatives for clean digestion
- Dam/Dcm methylation sensitivity reference showing which enzymes are blocked by E.coli methylation
- Type IIS enzyme guide (BsaI, BsmBI, BbsI) with cut distance specifications for Golden Gate/MoClo assembly
- T4 DNA Ligase protocols with temperature, timing, and vector-to-insert molar ratio recommendations for sticky and blunt ends
Frequently Asked Questions
What restriction enzymes are included in this database?
The database covers 15+ commonly used 6-cutter enzymes (EcoRI, BamHI, HindIII, XhoI, NdeI, KpnI, SalI, PstI, SpeI, NheI, BglII, EcoRV, SmaI), 8-cutter rare cutters (NotI with 8 bp recognition cutting every ~65,536 bp, PacI, SwaI), and special enzymes including DpnI (methylation-dependent), Type IIS enzymes for Golden Gate assembly, nicking enzymes, and homing endonucleases. NEB catalog numbers are provided for each enzyme.
What is the difference between 5-prime overhang, 3-prime overhang, and blunt end cuts?
A 5-prime overhang (e.g., EcoRI G^AATTC) leaves a single-stranded extension on the 5-prime end, creating "sticky ends" that facilitate ligation. A 3-prime overhang (e.g., KpnI GGTAC^C, PstI) leaves extensions on the 3-prime end. Blunt end cuts (e.g., EcoRV GAT^ATC, SmaI CCC^GGG) cut both strands at the same position. Sticky ends ligate more efficiently than blunt ends.
Which restriction enzymes produce compatible cohesive ends?
Key compatible end pairs include: BamHI (G^GATCC) with BglII (A^GATCT) producing compatible 4-nt 5-prime overhangs; XhoI (C^TCGAG) with SalI (G^TCGAC); and SpeI (A^CTAGT) with NheI (G^CTAGC). Fragments cut with compatible enzymes can be ligated together, though the resulting hybrid site may not be re-cut by either original enzyme.
What is star activity and how do I prevent it?
Star activity is non-specific cleavage at sites similar but not identical to the recognition sequence. It is caused by high glycerol concentration (>5%), excess enzyme, long incubation (>4 hours), non-optimal buffer or pH, and organic solvents. Prevention strategies include using High-Fidelity (HF) versions of enzymes from NEB, using the minimum amount of enzyme needed, and keeping incubation times to 1 hour or less.
How does Dam/Dcm methylation affect restriction enzyme digestion?
E.coli Dam methylase modifies GATC to G(m6A)TC, and Dcm methylase modifies CCAGG/CCTGG to C(m5C). Several enzymes are blocked by Dam methylation (BclI, ClaI, MboI, XbaI) or Dcm methylation (EaeI, AvaII). If your target site is affected, prepare plasmid DNA from dam-/dcm- E.coli strains to ensure complete digestion.
What is CutSmart buffer and which enzymes work with it?
CutSmart is NEB universal buffer containing 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 ug/mL BSA at pH 7.9 (25 degrees C). Over 95% of NEB restriction enzymes are active in CutSmart buffer, making it ideal for double digestions where two enzymes need to work in the same reaction.
How do Type IIS enzymes work for Golden Gate assembly?
Type IIS enzymes (BsaI, BsmBI, BbsI) cut outside their recognition sequence at a defined distance, generating custom 4-nucleotide sticky ends. For example, BsaI cuts 1 nt downstream of GGTCTC. This property allows seamless, scarless assembly of multiple DNA fragments in a single reaction, forming the basis of Golden Gate and MoClo modular cloning systems.
What are the optimal conditions for T4 DNA Ligase?
For sticky-end ligation, incubate at 16 degrees C for 30 minutes to 1 hour. For blunt-end ligation, incubate at 16 degrees C for 2 hours to overnight. Use 1x T4 Ligase Buffer containing ATP. The recommended vector-to-insert molar ratio is 1:3 to 1:5. NEB Quick Ligase enables ligation at 25 degrees C in just 5 minutes for rapid cloning workflows.