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Key Concepts

Gibson Assembly is a relatively new method for assembling DNA fragments. In traditional cloning methods, different pieces of DNA are cut with compatible restriction enzymes and ligated together to form the desired plasmid. This process can be difficult because not all desired DNA pieces have the right restriction sites in the right places and it can be difficult to obtain a high enough concentration of DNA for cloning. Gibson Assembly allows us to bypass these potential roadblocks by using PCR products with customizable junctions to make the exact plasmid we want, everytime.

Here, we will use Gibson Assembly to insert a sequence (GFP) into a linear vector (pET28b).  Gibson Assembly uses a one-pot reaction with an exonuclease, a polymerase, and a ligase. Our original GFP PCR was amplified using primers that added nucleotides on either side of the gene that are complementary to sequences flanking the BamHI cut site in pET28b. The three enzymes work together to construct our new plasmid:

  1. The exonuclease chews back nucleotides from the ends of the DNA fragments and creates a single-stranded 3´ overhang. Thus overhang facilitates the annealing of fragments that share complementarity.
  2. The polymerase fills in gaps within each annealed fragment. 
  3. The ligase seals nicks in the assembled DNA. 

After the assembly reaction is complete, a newly circularized construct containing the GFP sequence will be transformed into E. coli.


  • PCR tube
  • GFP insert (GFP PCR product after gel extraction)
  • Cut pET28b vector (BamHI-digested pET28b after PCR cleanup)
  • T4 DNA Ligase
  • 2X Gibson Master mix


Gibson Assembly Reaction Mix


Stock concentration Final reaction concentration

Amount to use (uL)

Gibson Master mix




Digested pET28b vector


50 ng


GFP insert


2-3 fold molar excess


T4 DNA ligase

400 U/uL

20 U


Nuclease-free water


Total Reaction Volume



20 µL


Calculation Help for Gibson Assembly Reaction:

  • 50 ng cut vector. Calculate the volume of vector you will need to add to your mixture to have 50 ng total by using the concentration of your digested vector from the Nanodrop.
  • 2-3 fold molar excess of GFP insert (732bp). Calculate the molar equivalent with the following formula, then multiply it by 2 or 3 depending on the available volume in your Gibson reaction. (pET28b is 5369bp)

Molar equivalent amount of insert (ng) = [Amount of vector (ng) x Size of insert (bp)] / Size of vector (bp)

  • Multiply this molar equivalent by 3, then using the concentration of your gel extracted insert, calculate the volume of insert you need to add to your mixture. (If the volume of your insert here plus the volume of your vector above is greater than 10 μL, only add 2 fold molar excess.)

Procedure for Gibson Assembly:

Important Note: Gibson Master Mix contains several sensitive enzymes that must be kept cold until the reaction is started on the thermocycler. Make sure you keep the Master Mix and the Assembly Mixture on ice at all times. The Master Mix is viscous, so pipette the liquid slowly and wipe the tip within the tube before adding it to the Assembly Reaction.

  1. Add the appropriate amount of your digested vector, GFP insert, and nuclease-free water (if necessary) to the PCR tube containing the thawed Gibson Master Mix in the order listed above. Be sure to slowly pipet up and down to mix just before placing the reaction in the thermocycler (or water bath). 
  2. Incubate the reaction at 50°C for 1 hour.
  3. Store the assembled plasmid at 4°C. The plasmid can now be used for transformation of E. coli.