Restriction enzyme digest map

Restriction digest analysis is essential when trying to clone a fragment of DNA into a plasmid. Typically, you would include restriction sites in the forward and reverse PCR primers used to amplify the insert you aim to clone. It is essential that you do not already have one or more of those recognition sites within the main sequence of your fragment; otherwise the enzyme will cut the DNA into two or more pieces instead of leaving a single fragment with sticky ends for cloning.

This tool maps common restriction enzyme recognition sites on a linear DNA sequence. Paste your sequence below to list enzymes that cut it (with 5′ positions on the forward strand) and to filter by how many times each enzyme cuts. Importantly, you can also list enzymes that do not cut your sequence. If you are adding specific sites on your PCR primers, those enzymes should appear in the zero cutters list — that check is essential before you begin cloning.

Be careful that the DNA you analyse does not carry epigenetic or chemical modifications (e.g. methylated bases), which can block some enzymes even when the recognition sequence is present. See NEB: Dam, Dcm, and CpG methylation for methylation-sensitive enzymes.

How to use this tool

1. Paste your DNA sequence (5′→3′) into the sequence box. Use A, T, G, and C only.
2. Choose an enzyme filter: all enzymes, zero cutters, 1 cutter, or 2 cutters.
3. Click Analyze. Read positions and fragment logic for your planned digests.
4. For a double digest, check buffer compatibility between the two enzymes. Many newer preparations (e.g. the NEB HF / rCutSmart range) share a single buffer; still confirm temperature preferences for each enzyme.

Cutting near DNA ends

Many restriction enzymes cut poorly when their site sits at the very end of a fragment, partly because the duplex can breathe or fray at the termini. For efficient digestion of PCR products, add a short clamp of extra bases beyond the recognition site. Different enzymes prefer different overhang lengths; rarely more than three extra bases are needed, so adding three clamp bases is a practical default. Read NEB: cleavage close to the end of DNA fragments.

Isoschizomers and compatible sticky ends

Different enzymes that recognize the same sequence are isoschizomers. For cloning, enzymes with different sites but compatible cohesive ends are often more useful. For example, XhoI cuts C/TCGAG (5′ overhang TCGA / 3′ AGCT), while SalI cuts G/TCGAC and leaves the same overhangs, so the sticky ends can ligate together — but the combined product destroys both the XhoI and SalI sites. See NEB: compatible cohesive ends.

Results list each enzyme’s recognition sequence and cut positions numbered from the 5′ end of your pasted strand. Use zero cutters when you need enzymes that do not cut internally, or filter for single or double cutters when planning a digest.

Related resources on The Bond Lab

• CloneIT2 — plasmid maps with restriction sites on a circular map.
• Protocols — lab methods, including pouring LB agar plates for cloning workflows.
• Primer design guide — planning sites in PCR primers.

Data & privacy

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