tooluniverse-molecular-cloning
Installation
SKILL.md
Molecular Cloning Assembly Design (Gibson & Golden Gate)
Plan how to join DNA fragments into a construct: design the overlaps (Gibson) or Type IIS overhangs (Golden Gate) and avoid the failures that come from internal sites and non-unique junctions.
Step 0 — Pick the method
| Use Gibson Assembly when | Use Golden Gate when |
|---|---|
| A few fragments, scarless/seamless junctions anywhere you choose | Many parts, standardized reusable parts (MoClo/modular), one-pot |
| You can add ~20–40 bp homology by PCR | You can remove internal BsaI/BbsI sites (domestication) |
| One-off constructs | Combinatorial libraries / repeated assemblies |
Both are sequence-independent (no scar at the junction for Gibson; a 4-bp fusion scar for Golden Gate). For 2–4 unique fragments, Gibson is usually simplest; for libraries or a parts toolkit, Golden Gate.