Gibson Assembly
Gibson Assembly, also known as enzymatic or chew-back assembly, is a powerful tool used for DNA cloning. It allows you to manipulate almost any fragment of DNA in any location, alleviating the need to plan projects around restriction sites.
Overview and Background Info
Gibson Assembly uses sequence homology between fragments of DNA to join them together. A Gibson Assembly reaction mixture uses 3 enzymes: 5'-->3' exonuclease, high-fidelity polymerase and ligase. Together, these enzymes chew back the 5' end of DNA fragments (allowing fragments with sequence homology to anneal), fill in any gaps created by the chew-back, and seal the nicks in the annealed fragment. While it can be used to generate linear assemblies, Gibson is most commonly used to manipulate circular plasmids, for transformation and propagation in bacteria. Fragments for Gibson Assembly must have 15-500 bp of homology. This can be done through synthesis of entire fragments, or by amplification with "overhanging" primers
Protocol
1. Prepare DNA required for the reaction, typically linearize vector (see restriction digest) and PCR amplification of insert(s) (see PCR)
2. In a PCR tube, prepare reaction mix as indicated below
| 2-3 Fragments | 4-6 Fragments | |
| Recommended DNA Molar Ratio (Vector:Insert) | 1:2 | 1:1 |
| Starting amount of vector | 50 – 100 ng | 50 – 100 ng |
| Amount of insert | depends on molar ratio | |
| 2X HiFi DNA Assembly Master Mix | 10 µL | 10 µL |
| ddH2O | x (up to 20 uL) | x (up to 20 uL) |
3. Incubate the sample in a thermocycler at 50°C for 15-30 mins for 2-3 fragments or 60 mins for 4-6 fragments
4. Transform into component E. coli
Reference
NEBuilder HiFi DNA Assembly Master Mix Instruction Manual