Drybench Plasmid.
Diagnostic restriction digest picker with a schematic gel
Pick the digest whose bands tell a correct, empty and reversed construct apart, and see the schematic gel before you run it.
The worked example: EGFP's coding sequence (720 bp, NCBI U55762.1) ligated into pUC19's SmaI site. The picker compares it with the empty vector and with the insert reversed, and lists the digests whose schematic gels tell all three apart.
Judged on unmethylated DNA (a PCR product or synthetic DNA), as the picker's reference check was; in the map, set the methylation of the DNA you will digest.
1,710 of 4,157 digests from a pool of 95 enzymes tell all three apart; the first are listed.
| Digest | Correct (bp) | Empty (bp) | Reversed (bp) |
|---|---|---|---|
| AcuI | 1,263, 1,012, 888, 199 | 1,674, 1,012 | 1,092 + 1,059, 1,012, 199 |
| EaeI | 1,442, 987, 490, 389 | 1,442, 987, 257 | 1,442, 987, 389, 307, 281 |
| CviQI | 1,773, 676, 434, 282, 241 | 1,769, 676, 241 | 2,195, 676, 282, 241 |
| RsaI | 1,773, 676, 434, 282, 241 | 1,769, 676, 241 | 2,195, 676, 282, 241 |
| Tsp45I | 1,589, 576, 489, 311, 230, 211 | 1,588, 576, 311, 211 | 1,729, 576, 489, 311, 211 |
| BmrI | 1,439, 1,306, 661 | 1,380, 1,306 | 1,930, 1,306, 170 |
| BtsI | 1,507, 1,195, 684 | 1,507, 1,159 | 1,507 + 1,489, 390 |
| BsiHKAI | 1,161, 820, 618, 497, 225 | 1,161, 718, 497, 225 | 1,316, 1,161, 497, 225, 122 |
Schematic: band order and log-size spacing on one scale for every lane, nothing more. No band has a millimetre or Rf position, no sample band is sized from the ladder or from another lane, no band is said to match a ladder band, and no agarose percentage is assumed. Fragments under 100 bp are not drawn. A fragment joins a band when it is within 5 percent of that band's largest fragment (a judgement), so the fragments drawn as one band are within 5 percent of each other and may not resolve. Uncut DNA is not drawn: its supercoiled, nicked and linear forms are not modelled.
Computed when this page was built, from data release f1.e1.p1.
How it is computed
- Diagnostic digest picker. Single and double digests compared on the plasmid as given, with the insert removed (empty) and with the insert reversed; a digest is offered when the schematic gel tells all three apart and cuts each of them; the verdicts are read from bands grouped under gel rule 2.0.0; the pool and the ranking are this app's judgement.Checked against the oracle's verdicts on the correct, empty and reversed trio (EGFP in pUC19's SmaI site both ways, and pUC19 empty) under gel rule 2.0.0 (trio v2, 2026-10-17): the pool of 95 enzymes and all 4,157 digests judged agree, bands included, 1,710 of which tell all three apart (unmethylated DNA). 43 of 4,157 digests of the EGFP-in-pUC19 trio change their bands under gel rule 2.0.0; no picker verdict on that trio changes, and the digests telling all three apart are the same set as under gel rule 1.0.0 (trio v1, 2026-10-06).
- Digest fragments. Fragments between consecutive top-strand cuts, across the origin of a circular plasmid; lengths in base pairs on the top strand, as Biopython and NEBcutter report them.Fragments checked against Biopython 1.88 Bio.Restriction on 446 single digests and 6 double digests of pUC19 and pBR322, lengths exact (2026-10-06).
- Schematic gel. Band order and spacing by the logarithm of size only; no millimetre or Rf positions, since no calibrated mobility model is used. Fragments under 100 bp are not drawn. Sizes taken largest first, a fragment joins a band only if it is within 5 percent (inclusive, a judgement) of that band's largest fragment, so a band's fragments are within 5 percent of each other; they may not resolve and are drawn as one. Two lanes are compared band by band through each band's largest fragment. Supercoiled, nicked and linear forms of uncut DNA are not modelled.Grouping checked against 11 hand-worked cases with exact fractions (gel rules 2), order and log spacing unit-tested (2026-10-17); no oracle exists for band positions, so none is drawn.
- Restriction sites and cut positions. Recognition sequences and cut positions as published facts, typed by hand (pipeline/plasmid-map/enzymes.csv); every row checked against Biopython 1.88 Bio.Restriction, whose data are REBASE release 404 (Roberts RJ et al. REBASE: a database for DNA restriction and modification. Nucleic Acids Res 2023;51:D629-D630).Every row checked against Biopython 1.88 Bio.Restriction (enzyme data release 404) in its own notation, overhang and cut positions: 223 enzymes on pUC19 and pBR322, 446 cases, cut positions exact, with sites across the origin and 6 double digests (2026-10-06).
Limits
- For research and education only, and not medical advice: plasmid maps and sequence assembly designs. A designed product is not evidence that the reaction occurred; nothing here predicts expression or how well a reaction will work.
- The gel is schematic: band order and spacing by the logarithm of size only, no millimetre or Rf positions; fragments under 100 bp are not drawn; a band holds the fragments within 5 percent of its largest (a judgement), which may not resolve; the forms of uncut DNA are not modelled.
- Enzyme sites and cut positions are from a hand-curated table of 223 enzymes (version 1, built 2026-10-06); it may differ from a vendor's list, so check NEBcutter before a critical digest. Star activity, Type IIB and nicking enzymes are not modelled.