Team:Newcastle/Gibson Cloning
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# On ice, add 5 ul DNA to 15 ul thawed 1.33X Master Mix | # On ice, add 5 ul DNA to 15 ul thawed 1.33X Master Mix | ||
# Incubate at 50°C for 60 minutes | # Incubate at 50°C for 60 minutes | ||
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Transformation of E. coli may be done with 5-20 ul of the assembly mix depending upon the cells and method of transformation. | Transformation of E. coli may be done with 5-20 ul of the assembly mix depending upon the cells and method of transformation. |
Revision as of 09:31, 5 August 2010
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Contents |
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).
- On ice, add 5 ul DNA to 15 ul thawed 1.33X Master Mix
- 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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