Gibson Assembly® Protocol (E5510)

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Optimal Quantities

Assembly Protocol:

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 vector with 2-3 fold molar excess of each insert. Use 5-fold molar excess of any insert(s) less than 200 bp.To achieve optimal assembly efficiency using in 4-6 fragment assemblies, use a 1:1 molar ratio of each insert:vector.Total volume of unpurified PCR fragments in the 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.