Optimization Tips for NEBuilder® HiFi DNA Assembly and NEB® Gibson Assembly

Looking to assemble multiple DNA fragments in a single reaction? Here are some tips to keep in mind when planning your NEBuilder HiFi DNA Assembly or Gibson Assembly experiments.


Decide How You Want to Generate the Linearized Vector. You can Choose from the Following Methods:

  • Restriction enzyme digestion: good for large plasmids you don’t want to amplify; background may be higher if undigested vector is present.

  • PCR: achieves lower background versus restriction enzyme digestion, but is limited by the size of the vector. Typically, vectors up to 10 kb can be amplified; for amplicons greater than 10 kb, divide into 2 fragments.

Design the Primers

  • Use the NEBuilder Assembly Tool to design the primers and check the sequence of the final assembly. Primers will contain the overlap sequence. We recommend watching the tutorials before using the tool for the first time. There is one for restriction enzyme digestion and another for PCR. These and other videos can be found at NEBuilderHiFi.com.

  • Make sure the overlap is the correct length for the number of fragments in the assembly: Refer to the section below titled “Use the Correct Amount of DNA” for more details.

Column Purify the PCR Products

  • If you do not purify the PCR products, limit the unpurified PCR products to 20% of the reaction volume (4 µl for a standard 20 µl reaction).

  • If PCR produces a single band of the correct size and the yield is good, DNA purification is not necessary.

  • If PCR produces multiple products or a smear, it is best to optimize the PCR. If it is not possible to optimize, purify the products using gel extraction. Be careful, however, as gel extraction can introduce guanadine thiocynate (from the gel dissolving buffer) and can reduce the efficiency of the assembly reaction. To minimize this contamination, trim the gel slice so that a smaller amount of gel dissolving buffer is required. Due to the potential for residual guanidine salt being present in fragments isolated by gel-extraction, PCR or DNA column purification (NEB #T1030) is preferable to gel extraction (NEB #T1020).

Use the Correct Amount of DNA

  • Make sure you calculate the optimum ratio of insert(s):vector. If the ratio is not ideal, we recommend using NEBioCalculator to determine molar amounts. This tutorial describes the use of the NEBioCalculator web tool module that converts mass to, or from, moles to help plan an NEBuilder HiFi DNA Assembly reaction.

    For NEBuilder HiFi DNA Assembly:

    2-3 fragments: 15-20 nt overlaps, total DNA = 0.03-0.2 pmol, 2 fold molar excess of each insert:vector
    4-6 fragments: 20-30 nt overlaps, total DNA = 0.2-0.5 pmol, 1:1 molar ratio of each insert:vector

    For NEB Gibson Assembly:

    2-3 fragments: 15-25 nt overlaps, total DNA = 0.02-0.5 pmol, 2-3 fold molar excess of each insert:vector
    4-6 fragments: 20-80 nt overlaps, total DNA = 0.2-1.0 pmol, 1:1 molar ratio of each insert:vector

Perform a PCR Assay to Determine if the Assembly is Successful

  • Determine if the assembly works in vitro by amplifying the assembled product directly from the assembly reaction. Dilute 1 µl of the assembly reaction with 3 µl water then use 1 µl as a template in a 50 µl PCR. Use primers that anneal to the vector and amplify across the insert. Do not use primers that anneal across the assembly junction because this can lead to false positive results. If you can amplify the assembled product but cannot recover clones by transformation, then the problem is either with the transformation step, or the inability of the cells to maintain the transformed construct due to toxicity.

    Check the reaction conditions, DNA amounts, overlap sequences and perform the assembly control.

Always use High Competency Cells with a Transformation Efficiency of 108 -109 cfu/µg

  • We recommend NEB 5-alpha High Efficiency Competent E. coli (NEB #C2987) or NEB 10-beta High Efficiency Competent E. coli (NEB #C3019).

    More information can be found on NEBuilderHiFi.com.