Description Hieff Clone™ Universal II One Step Cloning Kit is a new generation homologous recombinant cloning kit. The carefully optimized 2nd generation 2× Hieff Clone™ Universal II Enzyme Premix combines the recombinant enzyme and the buffer required for the recombinant reaction with the addition of a unique recombinant enhancer to significantly improve the efficiency of recombinant cloning. The kit can be directed to clone PCR products to any site of any vector, compatible with unpurified PCR products, directly recovered PCR products, low concentration of rubber recovered products, this product can reassemble the homologous arm GC content of 30%-70% of the joint fragment. The vector was completely linearized, and homologous sequences of the end of the linearized vector of 15-25 bp were introduced into the 5 'end of the positive and reverse PCR primers of the inserted fragment, so that the 5' and 3 'ends of the PCR products of the inserted fragment had exactly the same sequence corresponding to the two ends of the linearized vector, respectively. Under the action of recombinant enzyme, the recombinant reaction of PCR product and linearized vector can be completed in as little as 5 min at 50℃. The positive rate of cloning can reach more than 95%. Features Simple: Seamlessly assemble and clone up to six DNA fragments in a single reaction. Flexible: Design guidelines allow assembly into any vector of your choice. Efficient: Efficient for ligation of one to six fragments. Application Rapid Cloning: It allows multiple overlapping DNA fragments to be seamlessly linked in a one-step, 5–50 minute isothermal reaction. Directed Cloning; Site-Directed Mutagenesis. Specifications Cloning Process Seamless cloning Control Positive control Segments Up to 6 fragments Product Type Seamless Cloning and Assembly Kit Components Components No. Name 10923ES05 10923ES20 10923ES50 10923-A 2 × Hieff CloneTM Universal II Enzyme Premix 50 μL 200 μL 500 μL 10923-B 500 bp control insert (25 ng/μL) 5 μL 5 μL 5 μL 10923-C pUC 19 control vector, linearized (50 ng/μL) 5 μL 5 μL 5 μL Figures Figure 1. A total of 10 ng low-input single fragment was cloned into a 10 kb plasmid. The results showed that 10923ES had better performance than competing products, with high connection efficiency, more colonies and a positive rate of 100%. A-B: Colony plate. The molar ratio of carrier (10 kb) to insert fragment (1 kb) was 1:2. C: Electrophoresis analysis of the insertion fragment, M: YEASEN 10505ES. Plasmid input 40 ng/6 μL, 50°C, 15 mins. Figure 2. Six fragments (a total length of 7.6 kb) was cloned into a 11.6 kb plasmid. The results showed that 10923ES had better performance than competing products, with more colonies and positive rate of 100%. A-B: Colony plate. The molar ratio of carrier (11.6 kb) to insert fragments was 1:2. C: Electrophoresis analysis of the insertion fragment, M: YEASEN 10505ES. Plasmid input：200 ng/20 μL, 50°C, 50 mins. Figure 3. A 4.5kb fragment was cloned into a 21 kb plasmid. A: Colony plate. The molar ratio of plasmid to insert fragment was 1:2. B: Electrophoresis analysis of the insertion fragment, M: YEASEN 10505ES. Plasmid input：200 ng/20 μL, 50°C, 40 mins. Storage This product should be stored at -25~-15℃ for 1 years.
