A restriction enzyme reference lists the recognition sequence, exact cut position, and overhang type produced by each common restriction endonuclease, along with practical details like recommended buffer and heat-inactivation temperature. Molecular biologists and students use this kind of lookup table when designing cloning strategies, picking compatible enzyme pairs for a double digest, or troubleshooting why a digestion didn't go as expected.
| Enzyme | Recognition Sequence (5′→3′) | Cut Site | Overhang | Overhang Seq | Heat Inact. | Buffer |
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How to Use the Restriction Enzyme Reference
Search: Type an enzyme name (e.g. EcoRI), recognition sequence (e.g. GAATTC), or overhang type (e.g. 5-prime) to filter instantly.
Filter buttons: Filter by overhang type — 5′ sticky ends, 3′ sticky ends, or blunt ends.
Click any row to see the full detail panel with recognition sequence diagram, compatible ends, methylation sensitivity, and protocol notes.
About Restriction Enzymes
Restriction endonucleases are bacterial enzymes that cut double-stranded DNA at or near specific recognition sequences. They are essential tools in molecular cloning, DNA mapping, and recombinant DNA technology.
5′ overhang (sticky): 5′—AATTC—3′ ← most common, e.g. EcoRI
3′—C —5′
3′ overhang (sticky): 5′—C —3′ ← e.g. KpnI, SacI
3′—CATGG—5′
Blunt ends: 5′—GG|CC—3′ ← e.g. SmaI, EcoRV
3′—CC|GG—5′
// ↑ = cut position on top strand
// Compatible (cohesive) ends ligate efficiently
Choosing the Right Enzyme
When planning a cloning experiment, choose enzymes that produce compatible ends for ligation. Check that the enzyme does not cut within your insert or vector sequence. Also verify that the enzyme is not blocked by Dam, Dcm, or CpG methylation if your DNA is from a methylation-positive strain.
Heat Inactivation
Many restriction enzymes can be inactivated by heating to 65°C or 80°C for 20 minutes, eliminating the need for column purification before ligation. Enzymes marked "No" or "65°C" in this table require gel or column cleanup before ligation.
When to Use This Reference
Reach for this table whenever you're designing a cloning strategy and need to confirm an enzyme's exact cut position, overhang, or buffer before ordering reagents or setting up a digest. It's especially useful for picking a double-digest pair with compatible buffer conditions, confirming whether a vector's multiple cloning site contains a unique cut site for your enzyme of choice, or checking methylation sensitivity before digesting plasmid DNA prepared from a standard Dam+/Dcm+ E. coli strain. It also doubles as a quick teaching aid for explaining sticky-end versus blunt-end ligation to new lab members.
Common Mistakes to Avoid
Assuming any two sticky ends will ligate together — only complementary or identical overhang sequences anneal efficiently; matching recognition sequences alone does not guarantee compatibility, so always check the overhang sequence column, not just the enzyme name. Ignoring methylation sensitivity — digesting plasmid DNA from a standard Dam+/Dcm+ cloning strain with a methylation-sensitive enzyme can produce partial or absent cutting that looks like a failed reaction rather than a methylation block. Skipping cleanup after a non-heat-inactivatable enzyme — if the enzyme can't be heat-killed, residual activity can re-cut your ligated product unless you gel-purify or column-clean the digest first. Mixing incompatible buffers in a double digest — combining two enzymes that need very different salt concentrations or temperatures can lead to star activity (non-specific cutting) or incomplete digestion at one of the two sites.
Frequently Asked Questions
What is the difference between a 5′ overhang, a 3′ overhang, and a blunt end?
A 5′ overhang leaves single-stranded bases protruding from the 5′ end, as with EcoRI or BamHI, and is generally the easiest type to ligate efficiently. A 3′ overhang protrudes from the 3′ end instead, as with KpnI or SacI. A blunt cut leaves no overhang, with both strands cut at the same position, as with SmaI or EcoRV. Sticky ends need a compatible complementary overhang to ligate efficiently, while blunt ends can ligate to any other blunt end, usually at lower efficiency.
How do I know if two restriction enzymes produce compatible ends for cloning?
Compare the overhang sequence, not the recognition sequence — enzymes like BamHI, BglII, and BclI all leave the same GATC-based 5′ overhang and can be ligated to one another despite cutting different recognition sites. Always confirm the chosen enzyme doesn't cut a second time elsewhere in your insert or vector before committing to a digest.
Why does Dam, Dcm, or CpG methylation matter when choosing a restriction enzyme?
Standard E. coli cloning strains methylate DNA at Dam (GATC), Dcm (CCWGG), or CpG motifs, and some enzymes can't cut when their site overlaps one of these methylated sequences. This can look like a failed digestion even though the recognition site is present. Check the methylation sensitivity column here, and if needed use a Dam-/Dcm- strain or an insensitive isoschizomer.
What does it mean if a restriction enzyme cannot be heat-inactivated?
Most enzymes can be permanently killed by heating to 65–80°C for 20 minutes, letting you move straight into ligation. Enzymes marked "No" in this reference stay active after heating and need column or gel cleanup first, or they risk re-cutting your ligated product.
Can I use two different restriction enzymes in the same digestion reaction?
Yes — a double digest is common, but both enzymes need a shared or compatible buffer and overlapping optimal temperature. Many modern enzymes work in a universal buffer like CutSmart and can be combined directly; enzymes with very different requirements may need sequential digestion with a cleanup step in between.