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Team:Newcastle/Gibson Cloning
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===Reaction buffer recipes=== | ===Reaction buffer recipes=== | ||
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| + | PCR products that came from a plasmid template need to be purified on a 1% agarose TAE gel (adjust the gel and well size to accommodate the entire PCR (Phusion) reaction. This procedure is generally a good idea to reduce unwanted assemblies from minor aberrant PCR products. The Qiagen MiniElute gel extraction kits have been successful for this purpose as the final volume/concentration is approximately right for the assembly protocol. | ||
===One step isothermal Assembly=== | ===One step isothermal Assembly=== | ||
Revision as of 09:33, 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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PCR product purification
PCR products that came from a plasmid template need to be purified on a 1% agarose TAE gel (adjust the gel and well size to accommodate the entire PCR (Phusion) reaction. This procedure is generally a good idea to reduce unwanted assemblies from minor aberrant PCR products. The Qiagen MiniElute gel extraction kits have been successful for this purpose as the final volume/concentration is approximately right for the assembly protocol.
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.
Go back to our Protocol List
   
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