Protocol from New England Biolabs

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1. Set up the following reaction on ice:



	Recommended Amount of Fragments Used for Assembly
	2-3 Fragment Assembly	4-6 Fragment Assembly	Positive Control**
Total Amount of Fragments	0.02–0.5 pmols* X μl	0.2–1 pmols* X μl	10 μl
Gibson Assembly Master Mix (2X)	10 μl	10 μl	10 μl
Deionized H2O	10-X μl	10-X μl	0
Total Volume	20 μL***	20 μL***	20 μL

* Optimized cloning efficiency is 50–100 ng of vectors with 2–3 fold of excess inserts. Use 5 times more of inserts if size is less than 200 bps. Total volume of unpurified PCR fragments in Gibson Assembly reaction should not exceed 20%.
** Control reagents are provided for 5 experiments.
*** If greater numbers of fragments are assembled, additional Gibson Assembly Master Mix may be required.


2. Incubate samples in a thermocycler at 50°C for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled. Following incubation, store samples on ice or at –20°C for subsequent transformation.
   
   Note: Extended incubation up to 60 minutes may help to improve assembly efficiency in some cases (for further details see FAQ section).


3. Transform NEB 5-alpha Competent E. coli cells (provided with the kit) with 2 μL of the assembly reaction, following the transformation protocol.

For plasmid isolation, the Promega Wizard Plus SV Minipreps DNA Purification System A1460 Miniprep protocol was used. For PCR product isolation, the QIAquick PCR Purification kit was used.