Smolke:Protocols/DNA assembly
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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.
Notes
- As of 2-17-2014 Taq ligase is already included in the Gibson mix aliquots, so no additional enzymes need be added
- Gene assembly protocol page updated and moved here:
