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Team:British Columbia/Protocols/GibsonAssembly
From 2012.igem.org
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This protocol closely follows the suggested protocol in the NEB Gibson Assembly guidebook. | This protocol closely follows the suggested protocol in the NEB Gibson Assembly guidebook. | ||
| - | 1. Using purified DNA, add 50-100ng plasmid and 2-3X molar concentration of the inserts, or 5X if using inserts of 200 bp or less. Add dH2O up to 10 uL. Set up a positive control using 10uL Gibson Assembly Positive Control. | + | 1. Using purified DNA, add 50-100ng plasmid and 2-3X molar concentration of the inserts, or 5X molar concentration if using inserts of 200 bp or less. Add dH2O up to 10 uL. Set up a positive control using 10uL Gibson Assembly Positive Control. |
Revision as of 06:56, 2 October 2012
This protocol closely follows the suggested protocol in the NEB Gibson Assembly guidebook.
1. Using purified DNA, add 50-100ng plasmid and 2-3X molar concentration of the inserts, or 5X molar concentration if using inserts of 200 bp or less. Add dH2O up to 10 uL. Set up a positive control using 10uL Gibson Assembly Positive Control.
2. Add 10 uL Gibson Assembly Master Mix to each product.
3. Incubate at 50°C for 1 hour.
4. Using 2uL product, transform into competent cells. Since the Gibson Assembly Master Mix contains PEG, avoid using PEG sensitive competent cells. The provided positive control results in an amp-resistant plasmid.
