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Assembling gene fragments using isothermal assembly

Learn how to use the Gibson Isothermal Assembly method to quickly combine gene fragments, such as IDT gBlocks Gene Fragments, into large constructs.

Apr 11, 2012

Revised/updated Oct 27, 2016

gBlocks® Gene Fragments

gBlocks Gene Fragments are high fidelity, double-stranded DNA (dsDNA) up to 3000 bp in length that can be custom designed for virtually any cloning application, and are uniquely suited for assembling and modifying larger constructs using the Gibson Assembly® method. gBlocks Gene Fragments provide flexibility for synthetic biology applications that is not currently available in any other product.

Gibson isothermal assembly

The Gibson isothermal assembly method is based on the technique described by Gibson et al. in Nature [1]. The method relies on the use of an enzyme mixture consisting of the mesophilic T5 exonuclease, a thermophilic ligase, and a high fidelity polymerase. Both the vector and the 5′ ends of the gBlocks fragments to be assembled are designed with 30 bp overlaps. Prior to assembly, the vector must be linearized by a separate restriction digestion or PCR amplification.

For assembly, all of the components are combined and allowed to react at 50°C, where the exonuclease begins to digest dsDNA from the 5′ ends. The exonuclease is rapidly inactivated by the high temperature, leaving complementary 3′ single-stranded DNA (ssDNA) ends that anneal. The polymerase then fills in any remaining ssDNA gaps and the ligase covalently joins the fragments together (See Figure 1 in the DECODED article, Isothermal assembly: Quick, easy gene construction).

Isothermal assembly of gBlocks Gene Fragments makes it easy to create or modify genes without the need for restriction sites in your insert or for conducting time-consuming, sequential, cloning reactions. For more information on gBlocks Gene Fragments, visit www.idtdna.com/gblocks.

References

  1. Gibson DG, Young L, et al. (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods, 6(5):343–345.

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