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Gibson isothermal in vitro DNA assembly

We now maintain a stock of "Gibson mix" for multi-fragment DNA assembly as an alternative to traditional cloning for the Smolke Lab.

The mix is stored in 7.5 ul aliquots in PCR tubes in the bottom of the metabolic engineering -20C freezer.

The 1x concentration of the mix is 10 ul. So to carry out an assembly, add a total volume of 2.5 ul of prepped, linear DNA to the mix and incubate at 50C for 30-60 minutes and then directly transform the mix into heat-shock competent E. coli and plate onto the appropriate selective solid medium.


Tips


Making the mix Adapted from Gibson Nature Protocols April 2009

This is 1.2 ml of Gibson mix - the tricky part is aliquoting out the 160 individual PCR tubes with 7.5 ul each. Find an aliquoting buddy!


Notes

Gene assembly