In the following experiment, In-Fusion Cloning was used to create a positive control for localization studies of YFP-tagged proteins in planta. Bypassing the usual task of subcloning that comes with traditional ligation methods, yellow fluorescent protein (YFP) was cloned directly into a plant binary vector, under the control of the cauliflower mosaic virus double 35S promoter (CaMV D35S). Positive clones were screened and identified by restriction mapping and sequencing. The time from experimental design to a finished, verified clone was reduced by two days compared with ligation-based methods.

The desired insert was PCR-amplified to incorporate 5' and 3' ends designed specifically for cloning into the destination vector (Figure 1). The PCR generated a single, distinct band of the correct size (Figure 2, left). This insert was cloned into the linearized expression vector with the In-Fusion enzyme mix, yielding correct clones in three out of three colonies tested, as determined by restriction digest mapping shown for one of these clones in Figure 2 (right). The final plasmid was ~9.5 kb in size and built in a single round of cloning.

Figure 1. Schematic of the finished clone.The final plasmid (~9.5 kb) is an expression vector designed for use in planta, with YFP cloned in-frame with the CaMV D35S promoter.

Figure 2. A single, distinct band was obtained and cloned into the linear vector with 100% accuracy. Left. PCR amplification of cloning insert. YFP (740 bp) was amplified using CloneAmp HiFi PCR premix. Primers introduced 5'- and 3'-cloning ends suitable for the In-Fusion Cloning reaction. Right. Restriction digest mapping of finished clone. Plasmid DNA was cut with NcoI and XbaI, showing bands of the predicted size for correct clones.

In-Fusion Cloning generated positive expression clones with just one cloning reaction, with a success rate of 100%. No subcloning was necessary, and the time from experimental design to a finished, verified clone was reduced by two days, when compared with ligation-based methods.

Yellow fluorescent protein (740 bp) was amplified via PCR with CloneAmp HiFi PCR Premix, using 15 nmol of template in a 50-µl reaction. In order to facilitate a successful In-Fusion reaction, primers included 5' and 3' cloning ends made to match the ends of the linearized plant binary vector (pFCG19). PCR conditions were programmed with a standard denaturation step at 95°C, an annealing step of 54°C for 30 sec, and an extension time of 1 min at 72°C, for a total of 35 cycles.

The insert PCR product was separated on a 0.5% agarose gel and purified using the provided NucleoSpin Gel and PCR Clean-Up kit. Linearization of pFCG19 was performed with NcoI and XbaI according to the manufacturer's instructions. The purified PCR insert was cloned into linearized pFCG19 with the In-Fusion HD Cloning Plus (discontinued, replaced with In-Fusion Snap Assembly Master Mix) enzyme mix and then transformed into the included Stellar Competent Cells. Both protocols followed the instructions in the user manual. Transformed cells were plated on LB-agar plates with kanamycin (50 µg/ml) and incubated at 37°C for 18 hr. Three colonies were picked for cultures and screened by restriction digest mapping with NcoI and XbaI, followed by complete confirmation via sequencing.