In the following experiment, In-Fusion Cloning was used to clone five different PCR fragments into a lentiviral vector for eukaryotic expression. This vector had only one restriction site suitable for subcloning, which made it difficult and time-consuming to perform PCR cloning using traditional ligation methods. The In-Fusion protocol used below made it possible to eliminate those constraints. Each insert fragment was amplified with the included high-fidelity polymerase and cloned directly into the final lentiviral vector. Colonies of each transformant were screened by restriction digest in order to identify positive clones. Final vectors were complete in just three days, with only two to three hours of hands-on time required per day.

I have not tried ligation-based cloning for any of my current cloning experiments, but I have done a lot before we tested the In-Fusion kit. The major differences between these two cloning methods in our lab are the additional TA cloning, transformation, minipreps, digestion, and ligation needed for ligation-based cloning, which means at least three more working days. Typically we needed to screen at least nine colonies for each cloning experiment, with 70–80% positive confirmed by restriction digest. In my experience, the In-Fusion method is very convenient, reliable, and time-saving."

—Jun Yang, University of Texas Medical Branch

Five unique inserts, ranging in size from ~400 bp to 1.6 kb, were PCR-amplified to incorporate specific 5' and 3' ends designed for cloning into the destination lentiviral vector (~8 kb). Each PCR reaction generated strong, specific bands under a single set of PCR conditions. The inserts were cloned into the linearized vector with In-Fusion enzymes and transformed into the provided competent cells with very high efficiency. Positive clones were identified by screening three to five colonies each with restriction digest (Figure 1).

  

Figure 1. Restriction digest of clones. Screening of transformant colonies was performed via XbaI/EcoRI restriction digest. Results show only one negative colony (E1) out of all 18 colonies resulting from five individual cloning reactions.

In-Fusion Cloning generated five positive lentiviral clones with a success rate of nearly 100%. PCR conditions did not need to be modified for each insert, and direct cloning into a large vector was highly efficient. The cloning locus was based on experimental preference, instead of being limited by available restriction sites. Identification of positive clones was done with minimal screening, indicating high accuracy. Final vectors were complete in just three days, with only two to three hours of hands-on time required per day.

The following inserts were amplified via PCR with CloneAmp HiFi PCR Premix:

•  RSV NS1 and RSV NS2 fragments (~400 bp each)
•  human IRF3, IRF7, and IRF7 mutant cDNAs (~1.4–1.6 kb each)

Takara Bio's Primer Design Tool was used to design amplification primers that included 5'- and 3'-cloning ends to match the ends of the linearized lentiviral vector, such that they facilitated a successful In-Fusion reaction. PCR conditions were programmed with a denaturation step at 98°C, an annealing step of 55°C for 15 seconds, and an extension time of 1 minute at 72°C, for a total of 30 cycles.The insert PCR products were analyzed on an agarose gel and purified using the provided NucleoSpin Gel and PCR Clean-Up kit. Linearization of the destination vector was performed with XbaI and EcoRI according to the manufacturer's instructions.The purified PCR inserts were cloned into the linearized lentiviral vector with the In-Fusion HD Cloning Plus (discontinued and replaced with In-Fusion Snap Assembly) enzyme mix and then transformed into the included Stellar Competent Cells. Both protocols followed the instructions in the user manual. Three to five colonies were screened by restriction digest mapping with XbaI and EcoRI.

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