In-Fusion Cloning kits allow ligation-independent, directional cloning of PCR products into any vector, at any site of linearization (Figure 1). The cloning reaction takes as little as 15 minutes and enables you to build even more complicated constructs in just one step (Chen et al. 2009, Lestini et al. 2013).
Figure 1. In-Fusion Cloning protocol.
How does In-Fusion technology compare with another cloning system?
At first glance, In-Fusion Cloning technology has much in common with a method developed by Gibson and colleagues (Gibson et al. 2009). Both systems:
Provide options that eliminate restriction digest steps
Have relatively fast, simple protocols with just a few steps and reagents
Are seamless—that is, they don't add extra bases between joined fragments
Allow for multiple DNA fragments to be cloned in a single reaction
However, as we've previously touched on, there are critical differences in workflow and performance between seamless cloning systems. In-Fusion technology has a faster protocol, provides lower background, and reliably demonstrates higher cloning accuracy—especially where more complex cloning projects and high-throughput workflows are concerned. Under more challenging conditions and shorter reaction times, Gibson's system demonstrates problematically high levels of background, something it has in common with more traditional ligation and TA cloning methods.
Results
Putting In-Fusion technology to the test
In-Fusion Cloning technology was put up against Gibson's method in side-by-side experiments. In all instances, the cloning vector used pUC19, linearized with BamHI. Each cloning system was tested under the recommended conditions of its own cloning protocol, and additional testing was done with Gibson's enzyme mix for multiple-insert cloning. Gibson's method states the incubation time should be increased from 15 minutes to 60 minutes for four-fragment (three-insert) assemblies. In an effort to make a more direct comparison with In-Fusion Cloning, this multiple-insert experiment with Gibson's enzyme mix was also run at the shorter In-Fusion Cloning reaction time. All cloning reactions were then transformed into Stellar Competent Cells, and 1/10 of each reaction was plated. Colony counts and clone sequence verification were used to evaluate the results of each reaction. Sequencing data is the most precise measurement of cloning accuracy, and thus gives a much more informative analysis than simply comparing the total number of colonies. Results are shown below for both multiple-insert (Table I) and single-insert cloning (Table II).
In-Fusion Cloning
Gibson's method (short incubation)
Gibson's method (long incubation)
Conditions
Incubate at 50°C for 15 min
Incubate at 50°C for 15 min
Incubate at 50°C for 60 min
Vector + inserts
89 colonies
111 colonies
392 colonies
Negative control (no insert)
1 colony
39 colonies
78 colonies
Cloning accuracy
100% (26/26 correct colonies)
19% (5/26 correct colonies)
73% (19/26 correct colonies)
Table I. Results for multiple-insert cloning. Three fragments (in addition to the linearized vector backbone of 2.7 kb) were used as inserts in each reaction: MBP (1.1 kb), PROF12 (0.7 kb), and AcGFP1 (0.7 kb). Total finished plasmid size was 5.2 kb.
In-Fusion Cloning
Gibson's method
Conditions
Incubate at 50°C for 15 min
Incubate at 50°C for 15 min
Vector + insert
635 colonies
401 colonies
Negative control (no insert)
1 colony
39 colonies
Cloning accuracy
100% (26/26 correct colonies)
96% (25/26 correct colonies)
Table II. Results for single-insert cloning. One fragment (MBP; 1.1 kb) was used as an insert in each reaction with a linearized vector backbone (2.7 kb). Total finished plasmid size was 3.8 kb.
Accuracy counts—get the right clone, every time
For single-insert reactions, In-Fusion technology showed the expected high level of cloning accuracy. Gibson's technology showed a comparable level of accuracy when using In-Fusion Cloning conditions. However, the number of colonies seen in the negative control for Gibson's method was far higher than with In-Fusion Cloning and points to a much more precise, reliable cloning mechanism when using the In-Fusion enzyme.
The background observed when using In-Fusion technology was consistently lower than that observed when using Gibson's method, regardless of the number of inserts or reaction conditions. This difference was especially striking with multiple-insert cloning, where the total number of colonies are generally reduced and false positives present a larger problem. In-Fusion Cloning's high accuracy shines under the demands of multiple-fragment cloning—the negative control reaction gave an exceptionally low colony count, and the cloning accuracy reached 100%. In contrast, Gibson's cloning method was found lacking whether it was performed using In-Fusion Cloning's conditions, or Gibson's recommended conditions (which take four times as long).
Conclusions
In-Fusion Cloning delivers where the competition falls short
While both cloning methods provided good accuracy with single-fragment cloning, In-Fusion technology was the clear winner in terms of background, speed, and overall accuracy—especially when more complicated cloning projects were considered. In providing such a high level of cloning accuracy, In-Fusion technology reveals that the real measure of success is not in sheer numbers of colonies, but instead in the number of correct, error-free colonies. Researchers should be able to expect the right clone every time, and In-Fusion Cloning makes that possible.
We have also observed that In-Fusion technology has an additional advantage over Gibson's method with regard to maximum vector size (46 kb cosmid vs 20.5 kb plasmid). This additional flexibility means one In-Fusion reaction can take the place of a packaging system, quickly and accurately assembling a large vector that would otherwise be too unwieldy for cloning.
In-Fusion bundles provide everything needed for a cloning experiment, including a high-fidelity PCR polymerase, the cloning master mix, competent cells, and a kit to treat PCR products prior to cloning. Where the competing technology provides the bare minimum, In-Fusion Cloning kits fully equip the researcher for all steps, ensuring a high out-of-the-box success rate and allowing a direct comparison between the price per reaction with the true total cost of the experiment.
References
Chen, C. G., Fabri, L. J., Wilson, M. J. & Panousis, C. One-step zero-background IgG reformatting of phage-displayed antibody fragments enabling rapid and high-throughput lead identification. Nucleic Acids Res.42, e26–e26 (2014).
Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods6, 343–345 (2009).
Lestini, R. et al. Intracellular dynamics of archaeal FANCM homologue Hef in response to halted DNA replication. Nucleic Acids Res.41, 10358–70 (2013).
