Cloning methods compared: which one when?
Cloning comes down to joining an insert into a vector at the right place and in the right orientation. Methods differ in what they need and what scar they leave, so choose by the vector and insert you have.
Try it with the tool
In the cloning helper, see restriction maps and a virtual digest gel, and generate restriction-tailed or Gibson overlap primers right away.
Open the cloning helper →1. At a glance
| Method | How it works | Scar | Multi-fragment | Strength | Watch out |
|---|---|---|---|---|---|
| Restriction + ligation | Ends cut by the same enzymes joined by ligase | Site remains | Hard | Cheap and familiar | The insert must lack those sites |
| Gibson · HiFi | An enzyme mix joins overlapping ends (20–30 nt) | None (you design it) | Easy (2–6 pieces) | No site constraints | Short pieces (<200 bp) and repeats are tricky |
| In-Fusion | Joins via 15-nt overlaps | None | Possible | Fast and simple | Requires the kit |
| Golden Gate | Type IIS enzymes (e.g. BsaI) cut outside their site, joining via 4-nt overhangs | 4-nt junction only | Very easy (10+ pieces) | One tube; order and direction fixed | Internal BsaI sites must be removed |
| TA · TOPO | A-tailed PCR product ligated into a T-vector | Vector sequence | No | No primer design, very fast | Random orientation |
| Gateway | Recombination between att sites | att sites (~25 nt) | Limited | Easy transfer to many expression vectors | Needs an entry clone first |
2. How to choose
- The vector MCS has two enzymes absent from the insert → restriction cloning is cheapest; use two different enzymes to fix orientation.
- Exact, scarless placement or 2+ fragments → Gibson/HiFi.
- Repeated modular assembly (promoter + gene + tag) → Golden Gate.
- Quickly store or sequence a PCR product → TA/TOPO.
- Moving one gene into many expression systems → Gateway.
3. Restriction cloning steps and numbers
- PCR the insert with restriction-site-tailed primers (4–6 extra bases + site)
- Cut vector and insert with the same two enzymes → optionally dephosphorylate the vector to reduce self-ligation
- Gel-purify the cut pieces
- Ligation: 50 ng vector with insert at a 1:3 vector:insert molar ratio (insert ng = vector ng × insert bp ÷ vector bp × 3)
- Transform → antibiotic plate → screen colonies
The reagent calculator handles the molar ratio; the cloning helper checks sites and designs primers.
4. Verifying clones — three levels
| Method | What it tells you |
|---|---|
| Colony PCR | Whether the insert is present (orientation too with one vector and one insert primer) |
| Diagnostic digest | Overall size and orientation — do the fragment sizes match? |
| Sanger sequencing | Every base — mutations and reading frame (always do this last) |
5. Common failures
- No colonies: competent-cell efficiency, wrong antibiotic, failed ligation — include a control (uncut vector transformation).
- Only empty vector: vector cut at one end only or self-ligated → check both enzymes cut, dephosphorylate, gel-purify.
- Enzyme fails to cut PCR ends: too few extra bases → add 4–6.
- Blocked by methylation: XbaI, ClaI and others can be blocked by dam/dcm → check the vendor table, use a dam⁻ strain.
- Gibson fails: overlap Tm too low or wrong fragment ratio → 20–30 nt overlaps, 2:1 insert:vector.
In the cloning helper, see restriction maps and a virtual digest gel, and generate restriction-tailed or Gibson overlap primers right away.
Open the cloning helper →