Gibson assembly
One-pot, scarless joining of 2–6 DNA fragments using overlapping ends (15–40 bp homology). Use NEBuilder HiFi (recommended) or NEB Gibson Assembly Master Mix.
Primer design rules
- Add 20–30 bp of homology to each junction. The forward primer of fragment B starts with the last 20 bp of fragment A (in sense orientation), then the annealing region of B.
- Keep Tm of the annealing portion ~60 °C. Total primer length usually 40–60 nt.
- For multi-fragment assemblies, use online tools (NEBuilder Assembly Tool, SnapGene's Gibson primer designer, j5).
Reaction setup
| Fragments | Insert : vector molar ratio | Total DNA |
|---|---|---|
| 2–3 fragments | 2 : 1 | 0.03–0.2 pmol |
| 4–6 fragments | 1 : 1 | 0.2–0.5 pmol |
Reaction (10 µL)
- 2× HiFi assembly master mix — 5 µL
- Fragments at desired ratio — up to 5 µL
- Nuclease-free H₂O — to 10 µL
Pmol calculator: pmol = (ng × 1,000) / (bp × 650). NEB has a free online tool.
Procedure
- Combine fragments and master mix on ice. For ≥ 4 fragments, scale to 20 µL.
- Incubate 50 °C, 15 min (2–3 fragments) or 50 °C, 60 min (4+ fragments).
- Place on ice. Transform 2 µL into 50 µL high-efficiency competent cells (NEB 5α, DH5α, Stbl3).
- Recover in SOC at 37 °C, 1 h with shaking.
- Plate 50 and 200 µL on LB+antibiotic plates. Incubate 37 °C overnight.
- Screen 4–8 colonies by colony PCR + Sanger or whole-plasmid sequencing.
Troubleshooting
- No colonies? Check for transformation efficiency with a control plasmid. Reduce DNA amount — too much DNA inhibits.
- All colonies are empty backbone? Increase insert : vector ratio to 4 : 1, dephosphorylate the backbone, or use a backbone PCR rather than digest (eliminates re-circularisation).
- Many colonies but none correct? Suspect mis-priming or repeats; reduce homology overlap, redesign primers, or sequence-verify the source DNA.
- Fragments < 200 bp give poor efficiency. Order them as gBlocks/eBlocks instead.