Before you start
- Design primers with 20–40 bp overlaps between neighbouring fragments.
- Linearise the vector by PCR or restriction digest; DpnI-treat PCR products made from plasmid templates.
Reagents and materials
Totals are for 2 assemblies (+10% extra). Per-assembly (20 µL) amounts are in the middle column.
| Reagent | Per assembly (20 µL) | Total |
|---|---|---|
| Linearised vector (50–100 ng) | 75 ng | 75 ng |
| Insert fragments (2–3-fold molar excess each)calculate per fragment from length | — | as calculated |
| 2× assembly master mix | 10 µL | 10 µL |
| Nuclease-free waterto 20 µL total | 1 µL | 1 µL |
Equipment
- Thermocycler (50 °C)
- High-fidelity polymerase for fragments
- PCR clean-up or gel extraction kit
Procedure
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Prepare fragments
Amplify fragments with a high-fidelity polymerase and purify. DpnI-digest templates from plasmids for 15–60 min.
Calculate amounts
Use 50–100 ng vector and a 2–3-fold molar excess of each insert (5-fold for very small fragments).
Assemble
Mix fragments and vector with an equal volume of 2× master mix (total 20 µL).
Incubate
Incubate at 50 °C for 15 min (2–3 fragments) or up to 60 min (4–6 fragments).
Transform
Transform 2 µL into high-efficiency competent cells.
Screen
Colony PCR or diagnostic digest, then sequence the junctions.
Critical points
- Keep total DNA volume under ~20% of the reaction volume.
- Avoid strong secondary structure or repeats in the overlap regions.
Troubleshooting
| Problem | What to check or change |
|---|---|
| Few colonies | Check fragment amounts and overlaps, extend incubation, use higher-efficiency cells. |
| Template plasmid colonies | DpnI-digest and gel-purify fragments. |
Safety
Standard laboratory care.
Further reading
- Gibson DG et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods (2009).
This is a general starting-point protocol written for SolvesEverything. Follow your institution’s approved protocols, safety rules and manufacturers’ instructions, and optimise for your system. Spotted an error? Tell us.