Double Digest Calculator: Buffer, Activity and Temperature for Two Enzymes
Pick two restriction enzymes and get the single buffer that runs both, each enzyme's activity in every buffer, and a straight answer on when to digest sequentially instead.
Choose two restriction enzymes and get the buffer to use, chosen on the worse of the two activities rather than the average - because the enzyme that under-performs is the one that leaves your plasmid uncut and your ligation full of background. The full activity table for every buffer is shown, not just the winner, so you can see how much you give up by using a buffer you already have on the bench. Incubation temperatures are checked separately from buffer compatibility, since two enzymes can share a buffer and still disagree by 10 degrees, and that is a different problem with a different fix. Where no single condition works well enough, the answer is a sequential digest with a cleanup in between, and the tool says so plainly instead of recommending a compromise buffer that half-cuts. Activity figures come from NEB's own current product pages, audited against them in 2026 rather than transcribed from an older compatibility chart.
| Buffer | EcoRI | BamHI | Worse of the two |
|---|---|---|---|
| rNEBuffer 2.1recommended | 100% | 100% | 100% |
| rNEBuffer 3.1 | 50% | 100% | 50% |
| rCutSmart | 50% | 100% | 50% |
| rNEBuffer 1.1 | 25% | 75% | 25% |
The buffer is chosen on the worse of the two activities, because that enzyme is the one that leaves your plasmid uncut. A dash means NEB does not publish a figure for that enzyme in that buffer, not that it is zero.
- EcoRI is prone to star activity; avoid long digests / excess units / glycerol >5% in a shared-buffer double digest.
- Check template methylation (EcoRI blocked by CpG); use methylation-free DNA (e.g., dam-/dcm- strain) if needed.
- BamHI is prone to star activity; avoid long digests / excess units / glycerol >5% in a shared-buffer double digest.
- BamHI is not heat-inactivable; use column cleanup between steps if doing a sequential digest.
Not checked here: whether these two enzymes actually cut your sequence, how far apart the sites are, star activity, methylation sensitivity, or whether the fragments you get will resolve on a gel. Use the restriction-sites and virtual-gel tools for those.
Design, edit, and verify complete constructsSeqStudio combines sequence editing and annotation, plasmid maps, primer/cloning/CRISPR design, Sanger verification, and GenBank/SnapGene files in one workspace.
How to use the Double Digest Buffer tool
- 1Pick your two enzymes from the list. Only enzymes with published reaction data are offered, since a recommendation without activity figures behind it would be a guess.
- 2Read the recommended buffer and the worse enzyme's activity in it - that figure, not the better one, is what decides whether the digest goes to completion.
- 3Check the temperature row. Enzymes that share a buffer but not an optimum need the lower temperature and a longer incubation, or a sequential digest.
- 4If the answer is sequential, cut with one enzyme in its own buffer, clean up on a column or beads, then cut with the second.
Frequently asked questions
Why is the buffer chosen on the worse enzyme's activity?
Because a double digest fails asymmetrically. If one enzyme runs at 100% and the other at 25%, the reaction is limited by the 25% enzyme: you get linearized plasmid instead of the two-cut fragment you wanted, and after ligation that shows up as a lawn of colonies carrying re-ligated vector. Averaging the two activities would hide exactly the enzyme that causes that, so the recommendation is made on the minimum and the minimum is what the headline figure reports.
What activity is good enough?
As a working rule, 75% or more for both enzymes is comfortable, 50-75% works with a longer incubation or more enzyme, and below 50% is where a sequential digest becomes the better use of an afternoon. The table colors the figures on those bands. These are practical thresholds rather than a vendor guarantee - a 50% enzyme cutting a supercoiled plasmid at a site near another feature can behave worse than the number suggests.
The enzymes share a buffer but the temperatures differ. What do I do?
Digest at the lower of the two temperatures for longer, or digest sequentially. An enzyme run below its optimum is slower but usually still specific; run above its optimum it can lose specificity or die. The tool reports the two temperatures separately from the buffer verdict precisely because they are different failure modes, and a shared buffer does not rescue a 10-degree gap.
Why does a dash appear instead of an activity figure?
Because NEB does not publish a figure for that enzyme in that buffer. A dash is not zero and should not be read as one - it means the number is unknown, so the tool declines to recommend that buffer rather than assuming a value. If the only dash-free option is a poor one, that is genuine information about how well characterized the pair is.
Does this tell me whether the enzymes cut my sequence?
No, and it deliberately stays out of that question. This answers the buffer and temperature question for a pair of enzymes; whether those enzymes have sites in your plasmid, how far apart they are, and whether the resulting fragments will resolve on a gel are separate questions answered by the restriction-sites, double-digest fragment and virtual-gel tools. Keeping them separate means the buffer answer does not require you to paste a sequence.
What about star activity and methylation?
Neither is modeled here. Some enzymes cut at degenerate sites under sub-optimal conditions - exactly the conditions a compromise buffer creates - and some are blocked by Dam or Dcm methylation in the strain you grew the plasmid in. Both are real reasons a digest with a good activity score still fails, and both are outside what a buffer table can tell you. Notes flagged in the results call them out where the reaction data records them.
Where do the numbers come from?
From NEB's current product pages for each enzyme, checked against them directly rather than copied from a legacy compatibility chart - an audit in 2026 found 57 corrections between the older chart values and the current published ones, including cases where the modern rNEBuffer figures differ from the classic four-buffer values people still quote. The high-fidelity and standard versions of the same enzyme are not interchangeable and are listed separately where both exist.
Can I check a whole plate of digests at once?
Yes — for a whole plate of planned digests, use the batch workflow rather than checking one pair at a time.
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