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	<area shape="rect" coords="40,800,255,860" href="https://2012.igem.org/Team:British_Columbia/Protocols/Biodesulfurization" />		<area shape="rect" coords="40,800,255,860" href="https://2012.igem.org/Team:British_Columbia/Protocols/Biodesulfurization" />
	</map>		</map>
		+
		+	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.
		+
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Revision as of 06:54, 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.