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.