![]() Ligation cloning lagged behind, especially with the multiple-insert reactions.įigure 1. In-Fusion Cloning was successful in all tested conditions, providing high cloning efficiencies for both single and multiple inserts. Positive clones were identified by restriction digest, and the number of positive clones obtained from ten randomly chosen clones was averaged for five independent experiments to determine cloning efficiency (Figure 2). In all cases, the final plasmid was ~6.8 kb. All inserts were PCR-amplified and purified, however, inserts for ligation cloning were digested with the appropriate restriction enzymes prior to purification.Ĭloning reactions with one, two, or three inserts were set up for each cloning method (see Figure 1 and Table II), with the two- and three-insert reactions designed to clone all DNA fragments simultaneously into an expression vector. For ligation cloning, inserts were designed with compatible restriction sites for adjacent fragments (insert/vector or insert/insert see Figures 1 and 3). "įor In-Fusion Cloning, inserts were designed with specific cloning ends that overlapped with adjacent DNA fragments (insert/vector or insert/insert see Figure 1). ![]() In conclusion, the In-Fusion HD Cloning Kit provided us with a higher cloning efficiency and faster results compared to traditional ligation based cloning for both single and multiple insert cloning. However, when the experiments were performed with In-Fusion Cloning, the process was highly efficient for both single- and multiple-insert reactions, and was completed in a shorter time with less handling. Colonies were screened by restriction digest, and cloning efficiency was determined as the number of positive clones obtained from ten randomly chosen colonies, averaged across five independent experiments.īy directly comparing the two cloning methods, they found that single-step multiple-fragment cloning with traditional T4 ligase was extremely difficult due to low cloning efficiency. Cloning experiments using each method were set up for one, two, or three inserts, respectively. In this study, two methods were tested side-by-side for cloning single and multiple inserts: In-Fusion Cloning and ligation-based cloning. Josep Clotet’s research group routinely purify recombinant chimeric proteins from S. cerevisiae the group was looking for a fast, easy way to successfully clone the chimeric proteins their work requires. ![]()
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