USER Cloning and USER Fusion: The Ideal Cloning Techniques for Small and Big Laboratories
The explosive development of the field of molecular biology has led to the need for simpler and more efficient cloning techniques. These requirements are elegantly met by the ligation-free cloning technique called USER cloning. USER cloning is suitable not only for everyday and high-throughput cloning but also for the one-step construction of complex DNA constructs, which can be achieved in a variant called USER fusion. In this chapter, we present a general protocol for converting any vector into a USER-compatible vector, together with protocols for both USER cloning and USER fusion.
Key wordsLigation-free cloning high-throughput cloning seamless DNA fusion multiple PCR fragment assembly uracil excision-based cloning artificial microRNA homologous recombination Gateway cloning
The development of the methods described herein was financially supported by the Danish Council for Independent Research, through a Technology and Production Sciences (FTP) research grant (J.nr. 274-05-0451). Lis B. Møller is thanked for excellent technical assistance, and both Bjarne G. Hansen and Morten H. H. Nørholm are thanked for constant fruitful collaborations in the development of USER cloning.
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