Successful DNA cloning is critical for DNA engineering workflows in medical, veterinary, and agricultural research. A single error in the finished construct could ruin downstream steps, and therefore, the initial screening process must locate correct clones. However, the process is costly and labor-intensive, especially when the cloning technique is inefficient and the resulting constructs are inaccurate.

In-Fusion Snap cloning offers distinct advantages over other commercially available seamless cloning kits, including various Gibson assembly-based methods (Table 1).

Here, we demonstrate superior performance of the In-Fusion Snap Assembly Master Mix over the GeneArt Gibson Assembly HiFi Master Mix in two particularly difficult experiments: multi-fragment cloning and large fragment insertion. The side-by-side comparison shows In-Fusion Snap cloning overcame the most daunting challenges, producing high colony counts and consistently accurate constructs.

In-Fusion Snap Assembly vs. GeneArt Gibson Assembly HiFi in multi-fragment cloning

We created a particularly demanding feat of gene assembly for the competing methods, requiring simultaneous insertion of five different DNA fragments, ranging from 405 to 1,005 bp.

As shown in Figure 1, In-Fusion Snap Assembly produced approximately ten times as many colonies as did GeneArt Gibson Assembly HiFi. The respective means were 581 colonies vs. 61. When the cloning accuracy was confirmed by sequence analysis, In-Fusion Snap Assembly exhibited 90% accuracy (nine positive colonies out of ten) while GeneArt Gibson Assembly HiFi exhibited only 20% accuracy (two positive colonies out of ten).

In-Fusion Snap Assembly vs. GeneArt Gibson Assembly HiFi in large-fragment cloning

The next challenge for the two competing methods was insertion of a large fragment—34.2 kb—into the 2.6 kb-pMET vector. Ten random colonies from each cloning method were checked for accuracy of the construct.

As shown in Figure 2, the colony counts were drastically different between the two master mixes. In-Fusion Snap Assembly produced a mean of 802 colonies while the mean for GeneArt Gibson Assembly HiFi was 21. When the cloning accuracy was confirmed by colony PCR, the In-Fusion Snap Assembly Master Mix exhibited 90% accuracy (nine positive colonies out of ten) while the GeneArt Gibson Assembly HiFi Master Mix exhibited 60% accuracy (six positive colonies out of ten).

Many trailblazing research projects of DNA engineering and drug discovery begin with humble DNA cloning as a tool-building step. Accomplishing this accurately on the first try allows the entire workflow to advance quickly into the next phase. However, sometimes the constructs are complicated, with multiple pieces to assemble. Desired DNA fragments may be of large size or have complex structure. The resulting colony screens can be labor-intensive and filled with do-overs and delays. In-Fusion Snap Assembly has the flexibility to avoid these pitfalls. It provides consistent success in every scenario, especially those with extra challenges.

In this study, In-Fusion Snap Assembly Master Mix outperformed GeneArt Gibson Assembly HiFi Master Mix through the toughest cloning techniques. In-Fusion Snap Assembly enabled cloning of multiple inserts simultaneously into one linearized vector with nearly all colonies showing 100% sequence accuracy. In-Fusion Snap Assembly also enabled cloning of a particularly unwieldy fragment into another vector with efficiency far superior to GeneArt Gibson Assembly HiFi and exhibited consistently high accuracy.

The robustness of In-Fusion Snap Assembly makes this method well-suited to diverse scenarios. From small-scale manual to high-throughput cloning platforms, each reaction can be expected to yield numerous colonies containing accurate constructs.

To get started with this exceptional seamless cloning solution, we offer a convenient In-Fusion Snap Assembly Starter Bundle with everything needed for a cloning workflow, including high-fidelity PCR polymerase, a NucleoSpin Gel and PCR Clean-up kit, In-Fusion Snap Assembly Master Mix, and high-efficiency competent cells.

Multi-fragment cloning

Five different DNA fragments (ranging in size from 405 to 1005 bp) were prepared by PCR amplification. The pUC19 vector (2.7 kb) was linearized with Hind III restriction digestion. PCR primers were designed using the In-Fusion Cloning Primer Design Tool.

The five purified DNA fragments were simultaneously cloned into the pUC19 vector using each seamless cloning method according to the manufacturers’ protocols. Each cloning reaction was performed in triplicate. The reactions were then transformed into competent cells recommended by the respective manufacturers.

Ten random colonies were chosen from the array of plates corresponding to each of the two cloning methods. These were analyzed by Sanger sequencing to determine the cloning accuracy. Sequences were required to be one hundred percent identical to the reference sequence in order to be counted as accurate.

Large-fragment cloning

Sequences homologous to the ends of the 34.2-kb fragment were added to both ends of the pMET vector (2.6 kb) as it was linearized by inverse PCR using the primers designed with the In-Fusion Cloning Primer Design Tool. The 34.2-kb DNA fragment was assembled with the pMET vector using each seamless cloning method according to manufacturers’ protocols. Each cloning reaction was performed in triplicate. The reactions were then transformed into competent cells recommended by the respective manufacturers.

Ten random colonies were chosen from the array of plates corresponding to each of the two cloning methods. These were analyzed by colony PCR to determine the cloning accuracy. Primers were provided by a manufacturer as a screening primer set yielding an approximately 4.5-kb PCR product. Successful assembly was confirmed by the presence of a PCR product of the expected size on the agarose gel.