Team:Newcastle/Gibson Cloning

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Contents 1 Gibson DNA assembly 1.1 Background 1.2 Reaction buffer recipes 1.3 One step isothermal Assembly

Gibson DNA assembly

Background

This is a one-step isothermal method for assembling overlapping DNA fragments. Please see Enzymatic assembly of DNA molecules up to several hundred kilobases, Gibson et al. (2009).

Reaction buffer recipes

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One step isothermal Assembly

For ~6 kb fragments, use 10-100 ng DNA of each DNA in 5 ul final volume. Scale accordingly for fragment sizes (molar ratios).

1. On ice, add 5 ul DNA to 15 ul thawed 1.33X Master Mix
2. Incubate at 50°C for 60 minutes

Transformation of E. coli may be done with 5-20 ul of the assembly mix depending upon the cells and method of transformation.

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