Golden Gate assembly
One-pot digestion + ligation using a Type IIS enzyme (BsaI, BsmBI/Esp3I, SapI). Best for modular, repeatable, multi-part assembly (MoClo, Loop, etc.).
Design rules
- Each fragment is flanked by a Type IIS recognition site that points inward, leaving a defined 4 bp (or 3 bp with SapI) overhang after digestion.
- Adjacent fragments share complementary overhangs that uniquely define their order.
- Internal Type IIS sites in your fragments must be removed (silent mutations or domestication).
- Use NEB's Golden Gate Assembly Tool to choose orthogonal junctions for ≥ 6-fragment assemblies.
Reaction setup (20 µL)
| Component | Amount |
|---|---|
| Each fragment / part | 50–100 fmol (~75 ng of a 2 kb fragment) |
| Destination vector | 50 fmol |
| 10× T4 DNA ligase buffer | 2 µL |
| BsaI-HFv2 or BsmBI-v2 (10 U/µL) | 1 µL |
| T4 DNA ligase (400 U/µL) | 1 µL |
| Nuclease-free H₂O | to 20 µL |
Or use NEB Golden Gate Assembly Kit (a single mix containing the enzyme + ligase).
Thermocycler program
| Step | Temp | Time | Cycles |
|---|---|---|---|
| Cut + ligate | 37 °C | 5 min | 25–30 |
| Ligate | 16 °C | 5 min | |
| Final ligation | 16 °C | 5 min | 1 |
| Heat inactivation | 60 °C | 5 min | 1 |
| Hold | 4 °C | ∞ | — |
Simple 1-pot 1-step "37 °C × 1 h" works for 2–3 fragments. Use the cycle program for 4+ fragments.
Procedure
- Run the cycle program above.
- Transform 2–5 µL into competent cells (DH5α or similar).
- Plate on selective LB plates. If your destination vector has a dropout marker (e.g., ccdB or RFP), incorrect colonies will be visibly different.
- Pick white/non-fluorescent colonies. Confirm by colony PCR + sequencing.
Tips
- Equimolar parts give the best efficiency. Quantify carefully.
- If efficiency is low, increase to 30 cycles or extend the 37 °C step to 10 min.
- Watch for "skipped" overhangs — re-check that all designed junctions are unique (Hamming distance ≥ 1) on both strands.