Gibson Assembly Cloning

Gibson Assembly was developed by Dr. Daniel Gibson and his colleagues at the J. Craig Venter Institute.¹² The method allows assembly of multiple DNA fragments, regardless of fragment length or end compatibility. The method has been successfully used to assemble oligonucleotides, DNA with varied overlaps (15–80 bp) and fragments hundreds of kilobases long and has been rapidly adopted by the synthetic biology community due to its ease-of-use, flexibility and suitability for large DNA constructs.

Contents

1. Materials
2. Protocol

1. Materials

• The Gibson assembly kit is sold by NEB (product #E2611).
• Gibson blocks can be created by restriction digesting plasmids, by PCR or by de novo synthesis.
• Synthetic dsDNA “gene-blocks” up to 500 bp long can be purchased from IDT.

Note: These blocks have ahigh error rate (10% per block). Use these blocks only if you have to and make sure to sequence your constructs after Gibson assembly

2. Protocol

1. Use the NEBuilder tool PDF or design oligos for PCR-amplification of Gibson blocks.
2. Follow instructions in the NEB Gibson Assembly manual to combine Gibson blocks.

Footnotes

1. Gibson DG et al. (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods 6:343–345. ↩

2. Gibson DG (2011). Enzymatic assembly of overlapping DNA fragments. Methods in Enzymology 498:349–361. ↩