What this is for: Full digest planning over a 94-enzyme database (HF/plain versions, buffer, temperature, methylation): unit math with a glycerol warning, double-digest compatibility, and cutting frequency.
The formula: units needed = DNA (µg) × units per µg; cutting probability from the recognition sequence and GC%
How to use:
- Pick Enzyme 1 from the list and choose its version, HF (High-Fidelity) or Plain — recognition site, buffer, incubation temperature, heat inactivation, methylation notes and typical stock fill in from the database. If the enzyme has no HF version, the page says so and the Plain data is used.
- Enter the DNA amount, DNA volume and reaction volume in Reaction Setup.
- Set units per µg (10 U/µg pre-filled) and the stock concentration on your tube, and read the enzyme volume to pipette.
- Optionally pick Enzyme 2 for a double digest and read the buffer-compatibility verdict in the Reaction Summary, together with the glycerol check.
- For cutting frequency, enter your DNA length and GC% — expected cuts and average fragment size are computed from Enzyme 1's actual recognition sequence.
Enzyme 1
Pick an enzyme and a version; its data fills in from the 94-enzyme database (NEB NEBuffer Performance Chart and product pages).
HF = High-Fidelity engineered version (rCutSmart buffer, reduced star activity)
HF version available?
Recognition site
Recognition sequence
End type
Overhang
Recommended buffer (for chosen version)
Incubation temperature (°C)
Heat inactivation
Dam methylation
Dcm methylation
CpG methylation
Activity of chosen version in rCutSmart (%)
Typical stock from table (U/µL)
Enzyme notes
Reaction Setup
µg
µL — volume your DNA solution adds; 0 if you are only planning enzyme/buffer amounts
µL
h — reference only; HF/Time-Saver enzymes cut in 5–15 min, 1 h is the classic default
Enzyme 1 — Amount
U/µg — 1 U digests 1 µg λ DNA in 1 h by definition; 5–10 U/µg is the usual plasmid overdigestion
U/µL — overwrite with the value on your tube (typical table value above)
Total units needed — enzyme 1 (U)
Enzyme 1 volume to pipette (µL)
Enzyme 2 — Optional (Double Digest)
Leave Enzyme 2 on “none” for a single digest.
HF version available? (enzyme 2)
Recommended buffer — enzyme 2
Incubation temperature — enzyme 2 (°C)
Heat inactivation — enzyme 2
Activity of chosen version in rCutSmart (%) — enzyme 2
Typical stock from table — enzyme 2 (U/µL)
Enzyme 2 notes
U/µg
U/µL
Total units needed — enzyme 2 (U)
Enzyme 2 volume to pipette (µL)
Reaction Summary
10× buffer volume (µL)
Total enzyme volume (µL)
Water to final volume (µL)
Volume check
Enzyme share of reaction (%)
Glycerol check
Buffer compatibility (double digest)
Cutting Frequency — Enzyme 1
Computed from the actual recognition sequence (degenerate bases counted) and your GC%.
bp — pre-loaded with λ phage DNA (48,502 bp); use your plasmid/genome size
% — λ DNA is ~50% GC; E. coli ~51%, human ~41%, Streptomyces ~72%
Recognition site length (bp)
Probability of the site at any position
Expected number of cut sites
Expected average fragment size (bp)
Reference: Enzyme Database
All 94 enzymes in the database — the same table as the workbook's Enzymes sheet (NEB data, checked November 2026). Buffer / temperature / heat inactivation are shown as plain / HF.
| Enzyme | Site | Ends | Buffer (plain / HF) | Temp °C (plain / HF) | Heat inactivation (plain / HF) |
|---|
Prefer to work offline or keep a copy? The download link is right below.
Download the Restriction Digestion Calculator (Enzyme Database) workbook (.xlsx)
Download the Restriction Digestion Calculator (Enzyme Database) workbook (.xlsx)
Notes & limits (from the workbook): Worked example pre-loaded: EcoRI-HF cutting 1 µg in 50 µL at 10 U/µg from a 20 U/µL stock → 10 U → 0.5 µL enzyme, 5 µL of 10× buffer, 44.5 µL water; on λ DNA (48,502 bp, 50% GC) EcoRI is expected ~11.8 times, one site per ~4,096 bp. Glycerol: enzyme stocks are ~50% glycerol — keep TOTAL enzyme volume at or under 10% of the reaction so glycerol stays under ~5%; above that, star (off-target) activity becomes likely, especially for non-HF enzymes. Buffer and methylation data are per enzyme AND per version — re-check them if you switch HF/Plain. Frequency estimates assume random sequence with your GC% and independent positions; real DNA deviates (CpG depletion, repeats, methylation), and a circular molecule gives as many fragments as cuts while a linear one gives cuts + 1 — for the actual sites in a real sequence, use NEBcutter. Buffer names are NEB's system (NEBuffer r1.1 / r2.1 / r3.1, rCutSmart); other suppliers' buffers are not interchangeable by name. Data sources: NEB NEBuffer Performance Chart and NEB product pages (neb.com), checked November 2026 — always confirm against the datasheet in your enzyme box before an important digest.