69992  OrientExpress™ Random Primer cDNA Synthesis Kit

The OrientExpress™ cDNA Synthesis and Cloning Systems are complete sets of reagents designed for rapid, efficient construction of cDNA libraries having inserts in a defined orientation. Six systems are available based on the following vectors: a bacteriophage lambda insertion vector, λSCREEN-1, and two bacteriophage T7 vectors, T7Select1-1 and T7Select10-3. Each vector is available as a kit designed for either random or oligo(dT) priming. The OrientExpress Random Primer System is based on a patented, directional random primer strategy. The OrientExpress Oligo(dT) System uses a modification of conventional oligo(dT) priming to achieve orientation-specific cloning between EcoR I and Hind III sites. Starting with high-quality poly(A)⁺ RNA, up to five cDNA libraries can be constructed with either system.

The strategies for directional random primed and oligo(dT) primed cDNA synthesis are shown below. Both priming methods utilize special EcoR I/Hind III Linkers that generate a Hind III site only when ligated to sequences having AA at the 3′-end. The proper 3′-end is specified by the Hind III Random primers or by the Oligo(dT) primer used for first strand synthesis. Digestion with EcoR I and Hind III produces cDNA with an EcoR I site on one end and a Hind III site on the other, corresponding to the 5′- and 3′-ends of the mRNA, respectively. Ligation into EcoR I/Hind III vector arms produces inserts having a "sense" orientation relative to upstream expression signals.

Both methods produce large libraries; however, random primed libraries provide more even sequence representation and offer the ability to modify average insert size by adjusting the ratio of primers to mRNA in the first strand reaction. This feature is useful for creating libraries of protein functional domains for screening by ligand binding assays. It should be noted that, unlike oligo(dT), random primers will efficiently prime any RNA present in the sample. Therefore, we recommend using highly purified poly(A)⁺ RNA for random primed library construction to minimize the occurrence of rRNA and other "junk" sequences in the library. The Straight A's™ mRNA Isolation System is designed for the preparation of poly(A)⁺ RNA suitable for random priming.





For rapid, size fractionation of DNA and removal of small molecules (<300bp) from DNA solutions, please consider the Mini Column Fractionation Kit (Cat. No. 69995).