Hieff Clone™ Universal II One Step Cloning Kit is a newly upgraded 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 Directed: Enables directional seamless cloning of 1 to 7 DNA fragments. Convenient: No restriction enzyme digestion required for fragments; multi-fragment joining can be completed in a single tube. Rapid: Ligation reactions can be finished in as fast as 5 minutes. Efficient: High colony yield with a positive clone rate of over 95%. Versatile: Compatible with any vector, accommodating both sticky and blunt ends; suitable for multi-fragment assembly, site-directed mutagenesis, and more. Extra-long: Capable of vector fragment ligation lengths exceeding 25 kb. Applications 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 Ligation Up to 7 fragments Product type DNA Assembly Components Components No. Name 10923ES05 (5 T) 10923ES20 (20 T) 10923ES50 (50 T) 10923ES76 (10×50 T) 10923-A 2 × Hieff Clone™ Universal II Enzyme Premix 50 μL 200 μL 500 μL 10×500 μL 10923-B 500 bp control insert (25 ng/μL) 5 μL 5 μL 5 μL 10×5 μL 10923-C pUC 19 control vector, linearized (50 ng/μL) 5 μL 5 μL 5 μL 10×5 μL Figures Figure 1. 10 ng low-input single fragments were connected in the non-resuscitation receptive state of the 6μL small system. 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: Clony plate. The molar ratio of carrier (10 kb) to insert fragment (1 kb) was 1:2. C: Electrophoresis map identified by insertion fragment PCR, M: YEASEN 10505ES. 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. Ligation of ultra long fragments. The results show that the number of colonies in 10923ES is high, with a positive rate of 100%.A: Recombinant transformation plate. The molar ratio of the vector (21 kb) to the insert fragment (4.5 kb) is 1:2.B: PCR identification electrophoresis of the insert fragment, M: Yeasen 10505ES. Vector amount: 200 ng in a 20 μL reaction system, reaction conditions: 50°C, 40 min. Figure 4. Efficient Recombination of Low-Concentration Multiple Fragments. Low-concentration five-fragment and six-fragment assemblies were performed respectively. The results demonstrate that 10923ES exhibits stable ligation performance at low concentrations, yielding a high number of colonies with a 100% positive rate. A–B: Recombinant transformation plates. The molar ratio of vector (11.6 kb) to insert fragments (total 5.6 kb for 5 fragments; total 7.6 kb for 6 fragments) was 1:2. C–D: Gel electrophoresis of colony PCR verification for inserted fragments. M: Yeasen 10505ES; colony PCR product size: 2.6 kb. Figure 5. Excellent Stability. Following storage at 37°C for 7 days, the results demonstrate that 10923ES still stably ligates five and six fragments at low concentrations. A–F: Recombinant transformation plates. The molar ratio of vector (11.6 kb) to insert fragments (total 5.6 kb for 5 fragments; total 7.6 kb for 6 fragments) was 1:2. G–H: Gel electrophoresis of colony PCR verification for inserted fragments. M: Yeasen 10505ES; colony PCR product size: 2.6 kb.
