As the first step in so many experiments, successful cloning is key to generating meaningful data. The cloning tools you use can be the difference between wasted effort and quick, accurate results. The products in the In‑Fusion Cloning portfolio enable insertion of any PCR fragment or multiple fragments into your linearized vector for fast, seamless cloning. The In‑Fusion Cloning reaction is specific and directional, ensuring an exceptionally high rate of cloning efficiency in all applications. Unlike transformation efficiency, which measures the number of transformed colonies on a plate, cloning efficiency is a measure of accuracy, providing information on the number of correct clones obtained from the cloning reaction. Our standards for accuracy involve more than simple transformation efficiency. Why waste time screening a large number of colonies when you can pick just a few colonies, generated with In‑Fusion technology, and be confident that you have the right clone?
The efficiency of your cloning experiment becomes even more vital when you need to create constructs from multiple fragments. Multiple-fragment cloning experiments pose additional challenges as each insert must be kept in frame in order to build complicated constructs, to stitch genes together from synthetic building blocks, or to engineer new functionalities through combinatorial protein domain swapping, etc.
We have recently taken this technology to the next level—enhancing both the transformation and the cloning efficiency of the In‑Fusion Cloning reaction—for multiple-insert applications.
The In‑Fusion Cloning reaction generates accurate, directional cloning of desired fragments
The key to the In‑Fusion Cloning reaction is its proprietary enzyme that generates short regions of single-stranded overlaps between vector and insert(s), facilitating accurate, directional cloning of the desired fragments. This overlap is designed into the PCR primers used to amplify the desired insert sequences.
Figure 1. The In‑Fusion Cloning workflow.
Optimized protocol improves cloning performance for multiple-insert experiments
Historically, the In‑Fusion Cloning reaction has required 15 bp of homologous overlap between adjacent fragments (e.g., vector and insert). While this 15-bp overlap provides excellent results for single-insert cloning reactions, our scientists have further optimized the protocol for multiple-insert cloning, and found that increasing the overlap to 20 bp in this application improved both the colony count and the cloning accuracy. We tested this change from 15 bp to 20 bp using both the liquid and lyophilized (EcoDry) formats of the In‑Fusion HD Cloning Plus products in the construction of a vector plus five inserts. The results showed a colony increase of 5.6X and 4.7X for the liquid and the lyophilized kits, respectively (Figure 2).
However, since colony count is only part of the performance equation, we also analyzed the accuracy obtained when using a 20-bp overlap protocol. Results indicate that our increase to a 20-bp homologous overlap improves upon the already highly accurate cloning results that we see with the 15-bp overlap, increasing to 90% and 100% for the liquid and lyophilized formats, respectively (Figure 2).
|Kit type||Colony count||Fold-change||Accuracy|
|15-bp overlap||20-bp overlap||15-bp overlap||20-bp overlap|
Figure 2. Colony count and cloning efficiency (accuracy) with 15- and 20-bp homologous overlaps. Five inserts (947 bp, 717 bp, 697 bp, 405 bp, and 1,005 bp) were cloned into a 2.7-kb vector using a 1:2 vector-to-insert molar ratio. For each EcoDry and liquid In‑Fusion reaction, a 1/10 dilution was plated. For each transformation, 20 clones were randomly screened by sequencing. Negative controls with the same fragments and vector produced minimal/no background.
In other cloning systems, increasing the number of fragments can have a detrimental effect on both transformation efficiency and cloning accuracy. However, with a newly optimized protocol, In‑Fusion Cloning portfolio ensures the same high level of accuracy you see when single-insert cloning, even when you are performing more challenging multiple-fragment cloning projects.
New tools available to assist you in achieving a successful cloning project, every time
As the number of inserts required in your cloning experiment increases, so does the importance of using the right primers. To assist you with your primer design, we have developed a new online tool to guide researchers through the process, step by step. The In‑Fusion Cloning Primer Design Tool allows for multiple-insert cloning, in addition to single-insert cloning and site-directed mutagenesis. Simply input the DNA sequences of your vector and multiple inserts along with your linearization method to automatically generate primers for your cloning experiment. This new tool provides both a final vector sequence and your primer sequences, enabling a seamless transition from design to bench work.
To coincide with the release of the optimized protocol and the new In‑Fusion Cloning Primer Design Tool, we have also updated our technical documentation to include new guidelines for multiple-insert cloning:In‑Fusion HD Cloning Plus (liquid)
With less time spent on experimental design and cloning, you can spend more time advancing your research. Check out our In‑Fusion Cloning selection guide to choose the right kit for you.