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	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 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.
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	2. Add 10 uL Gibson Assembly Master Mix to each product.		2. Add 10 uL Gibson Assembly Master Mix to each product.
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	3. Incubate at 50°C for 1 hour.		3. Incubate at 50°C for 1 hour.
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	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.		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.
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Revision as of 06:55, 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 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.