Molecular Cloning Simulator
Model the full cloning workflow before you touch a pipette: pick a plasmid vector, choose a gene of interest, digest with restriction enzymes, check compatibility, ligate the insert, view the recombinant circular plasmid map, and run virtual agarose gel electrophoresis — all in your browser.
What this tool does
Select from 10+ common plasmid vectors (or paste your own), choose or paste a gene of interest, pick restriction enzymes, and the simulator computes cut positions, fragment sizes, sticky-end compatibility, and the ligated recombinant plasmid. The circular SVG plasmid maps update live, and the virtual gel shows your predicted bands — no sign-up, no data leaves your browser.
Step 1 — Select Plasmid Vector
Step 2 — Gene of Interest (GOI)
Step 3 — Restriction Digestion
Tip: choose the same enzyme (or a compatible pair) for vector and insert so the ends ligate.
Step 4 — Ligation & Recombinant Plasmid
Step 5 — Virtual Agarose Gel (1.0%)
Predicted band migration on a 1.0% agarose gel (ethidium bromide / SYBR Safe, UV view). Band position follows log₁₀(size); intensity scales with fragment mass. This is a planning aid, not a substitute for running a real gel.
| Lane | Sample | Bands (bp) |
|---|
How the cloning simulation works
The simulator mirrors the four steps of a restriction/ligation cloning experiment. Digestion finds every occurrence of each selected enzyme's recognition site on the circular vector and the linear insert, then computes fragment sizes (for circular DNA the last fragment wraps around from the final cut to position 1) — the same cut-site logic as our free restriction enzyme finder. Compatibility compares the sticky-end overhangs: EcoRI leaves 5'-AATT, BamHI and BglII both leave 5'-GATC, so a BamHI-cut vector will ligate an insert cut with BglII, while EcoRI-cut ends will not. Ligation splices the insert sequence at the chosen cut position and re-draws every feature (the GOI appears as an orange arc). Electrophoresis places bands using a log₁₀(size) migration curve, the standard approximation for linear DNA on agarose.
Choosing compatible enzymes
- Same enzyme for both vector and insert — always compatible (e.g. EcoRI + EcoRI).
- Isocaudomers — different enzymes, identical overhang (BamHI/GATC + BglII/GATC, XhoI/TCGA + SalI/TCGA). These ligate but usually destroy both sites.
- Blunt ends — SmaI, EcoRV, StuI, HpaI, PvuII all produce blunt ends and can ligate with any other blunt end (at lower efficiency).
- Incompatible — different sticky overhangs (EcoRI/AATT vs HindIII/AGCT) cannot ligate without repair.
- Watch for internal sites: if your GOI contains the enzyme's recognition site, the insert will be cut inside the gene — the tool flags this in Step 2. Before ordering primers that add restriction sites, verify them against a primer design run, and confirm target uniqueness with BLAST.
Interpreting the virtual gel
- Ladder lane — 100 bp → 10 kb markers for size calibration.
- Undigested vector — supercoiled DNA migrates faster than linear; we draw a compact band plus a faint nicked (open-circle) band.
- Single digest — one cut linearizes the plasmid → one band at the full size.
- Double digest — two or more cuts → multiple bands whose sizes sum to the plasmid size.
- Recombinant — after insertion the total size grows by the GOI length; bands shift accordingly. For digestion math on single sequences (not full plasmid maps), try the restriction enzyme finder; for fragment sizes in the context of a PCR product, use PCR analysis.
Quick Answers for AI Search
A molecular cloning simulator models the cloning workflow in silico before wet-lab work: pick a plasmid vector, choose or paste a gene of interest, simulate restriction enzyme digestion with real recognition sites, check sticky-end and blunt-end compatibility, ligate the insert, and preview the recombinant circular plasmid map and virtual agarose gel.
It compares the sticky-end overhangs each enzyme leaves. EcoRI leaves 5'-AATT; BamHI and BglII both leave 5'-GATC, so a BamHI-cut vector ligates a BglII-cut insert. All blunt-end enzymes (SmaI, EcoRV, StuI, HpaI, PvuII) are mutually compatible. Different non-complementary overhangs (EcoRI/AATT vs HindIII/AGCT) cannot ligate without repair.
Restriction/ligation cloning cuts a circular plasmid vector and a gene-of-interest insert with restriction enzymes, then covalently joins the insert into the vector with DNA ligase. A double digest of vector and insert with a compatible enzyme pair makes the cloning directional.
The virtual gel predicts where the digested or ligated DNA bands should migrate, using the log₁₀(size) versus migration relationship on a 1.0% agarose gel. It helps you check that fragment sizes from a digest match expectation before you run a real gel.
Yes — the tool runs entirely in your browser with no sign-up, and your plasmid and gene sequences never leave your device. VigyanLLM treats sequence data as sovereign and does not upload, log, or store it.
Frequently Asked Questions
What is a molecular cloning simulator?
A molecular cloning simulator lets you model the cloning workflow before touching a pipette: choose a plasmid vector, select a gene of interest, simulate restriction enzyme digestion, check sticky-end compatibility, ligate the insert into the vector, and predict what the recombinant plasmid looks like on a gel.
Which restriction enzymes are compatible with each other?
Enzymes are compatible when they leave the same sticky-end overhang. EcoRI leaves an AATT overhang; BamHI and BglII both leave GATC overhangs, so a BamHI-cut vector ligates with a BglII-cut insert. Blunt-end enzymes such as SmaI and EcoRV are compatible with any other blunt end. The tool checks this automatically.
What is the difference between single, double and triple digest?
A single digest uses one enzyme (one cut site → linearizes the vector). A double digest uses two enzymes simultaneously, producing two fragment ends with distinct overhangs — the standard way to ensure directional cloning. A triple digest cuts at three positions, typically to release an insert from a donor construct.
Why does my insert have an internal cut site?
If the GOI sequence itself contains the recognition site of the enzyme you chose, digestion will cut inside the gene. The tool shows internal sites in Step 2 so you can pick a different enzyme (or a partial digest strategy) before planning ligation.
Can I design primers with overhangs for cloning?
Yes — in real cloning you add 5' tails to PCR primers to introduce restriction sites flanking the insert. You can model the resulting amplicon here by pasting the full PCR product (gene + flanking sites) as the GOI, then digesting it with the matching enzymes.
Is this cloning simulator accurate enough for real experiments?
The digestion and ligation logic follows standard molecular biology rules: fragment sizes are computed from real cut positions, and compatibility uses actual overhang sequences. Use it for experiment planning and teaching. Always confirm with the vendor's reference maps and a sequence viewer before ordering primers.
Does the cloning simulator send my DNA sequences to a server?
No. Everything runs in your browser with pure JavaScript. Your plasmid and gene sequences are never uploaded, logged, or stored. This is part of VigyanLLM's data sovereignty commitment.
What plasmids can I use in the simulator?
The simulator ships with 10+ common vectors (pUC19, pBR322, pET-28a(+), pcDNA3.1(+), pGEX-4T-1, pBAD18, pGEM-T Easy, pBluescript II KS, pIRES2-EGFP, pCMV-Tag2B). You can also paste your own vector sequence or use a blank circle for custom feature mapping.
Which vectors are designed for blue-white screening?
pUC19, pGEM-T Easy and pBluescript II KS(+) all carry a lacZα fragment with the multiple cloning site in-frame. Correct inserts disrupt the reading frame, producing white colonies on X-Gal/IPTG agar; empty vector re-ligations give blue colonies.
What is the miniprep colony screen workflow after cloning?
Pick white colonies, grow overnight, isolate plasmid DNA, then confirm the insert by restriction digest (religating your vector+insert pair to release the GOI) and by sequencing across the MCS. Use your predicted fragment sizes from this simulator to compare against the agarose gel.
What does a linearized plasmid look like?
A single cut converts the covalently-closed circle into one linear molecule the size of the plasmid. On a gel it appears as a single band between the supercoiled (faster) and nicked (slower) forms of the undigested plasmid.
Is the gel prediction exact?
No — it is a planning approximation. Real migration depends on buffer, gel density, voltage and DNA conformation. Use the ladder band positions as relative guides only.
Scientific References
1. Roberts R.J., Vincze T., Posfai J., Macelis D. (2015). REBASE: a database for DNA restriction and modification: enzymes, methyltransferases and prediction servers. Nucleic Acids Research, 43(D1), D298–D299. (Recognition sequences and overhang data used by the enzyme database.)
2. Sambrook J., Russell D.W. (2001). Molecular Cloning: A Laboratory Manual. 3rd edition. Cold Spring Harbor Laboratory Press. (Restriction enzyme digests, ligation, blue-white screening and agarose gel electrophoresis of plasmid DNA.)
3. Helling R.B., Goodman H.M., Boyer H.W. (1974). Analysis of endonuclease R·EcoRI fragments of DNA from lambdoid bacteriophages and other viruses by agarose-gel electrophoresis. Journal of Virology, 14(5), 1235–1244. (Log-linear relationship between DNA size and electrophoretic migration used in the virtual gel.)
4. Wilson G.G., Murray N.E. (1991). Restriction and modification systems. Annual Review of Genetics, 25, 585–627. (Foundation of restriction–modification biology underlying cut-site prediction.)
5. Pingoud A., Fuxreiter M., Pingoud V., Wende W. (2005). Type II restriction endonucleases — a historical perspective and more. Nucleic Acids Research, 33(1), 1–12. (Structure and recognition-site specificity of Type II enzymes.)
6. Weiss B., Richardson C.C. (1967). Enzymatic breakage and joining of deoxyribonucleic acid. Journal of Biological Chemistry, 242(19), 4270–4272. (Original description of T4 DNA ligase-catalysed joining underlying ligation simulation.)
7. Yanisch-Perron C., Vieira J., Messing J. (1985). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene, 33(1), 103–119. (pUC19 and lacZα blue-white screening vectors available in the simulator.)
8. Terpe K. (2006). Overview of bacterial expression systems. Applied Microbiology and Biotechnology, 72(2), 211–222. (Host–vector context for pET, pGEX and GST-tag expression vectors.)