Superscript® Full Length cDNA Library Construction Kit
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Note: SuperScript® Full-Length cDNA Library Construction Kit is now available with new and improved primers and adaptor. Portions of the sequences for each oligonucleotide have been removed for more robust BP recombination reaction efficiency and increased colony forming units. More colonies means improved library complexity with less starting material required.
The SuperScript® Full-Length cDNA Library Construction Kit has been developed for generating full-length cDNA libraries. The kit incorporates an antibody-based enrichment step in a simple, robust protocol, and utilizes a 5-CAP antibody capped to magnetic beads to prevent truncated mRNA from being reverse-transcribed during cDNA synthesis.
This unique, full-length library construction technology combines SuperScript® III Reverse Transcriptase with Gateway® cloning technology, enabling recovery of previously unobtainable, full-length transcriptome sequences. The kit also avoids the use of time-consuming, inefficient ligation reactions, making library construction faster and offering better cDNA representation.
The SuperScript® Full-Length cDNA Library Construction Kit ensures: • A simplified, easy to follow protocol without the complicated steps of CAP trapper cDNA library construction methods (see figure 1) • Creation of a cDNA library that represents full-length-selected transcripts from total RNA or enriched mRNA (98–100% full length clones) • Highly efficient cloning of cDNA into multiple destination vectors without the need of restriction enzyme digestion and ligation
How the SuperScript® Full-Length cDNA Library Construction Kit Works
• Full-length enrichment through the CAP-antibody bead: Without a 5' CAP-based full-length enrichment of mRNA, at least a third of synthesized cDNAs do not contain full-length open reading frames. Chemical enrichment methods are time consuming and not robust. The SuperScript® Full Length cDNA Library Construction Kit is the only kit on the market with a CAP-antibody that directly binds the CAP structure of the mRNA with high affinity, allowing the recovery of 98–100% full-length cDNAs.
• High yields of cDNA: The SuperScript® Full-Length cDNA Library Construction Kit contains SuperScript® III Reverse Transcriptase (RT) for generating high cDNA yields. SuperScript® III Reverse Transcriptase, a proprietary mutant of SuperScript® II RT, is active at 50°C and has a half-life of 220 minutes. It also has a point mutation that reduces RNase H activity, thereby decreasing RNA degradation during first-strand synthesis.
• Gateway® cloning technology avoids restriction enzyme cloning: Library construction is mediated by Gateway® cloning technology, a site-specific recombination system that eliminates the use of restriction enzymes and ligase in cloning. Each cDNA insert is flanked by specific att recombination sites (added during the cDNA synthesis steps) that recombine with complementary att sites present in Gateway® donor vectors to create entry clones. Entry clones are subsequently recombined with Gateway® expression vectors to create expression clones, eliminating the use of restriction enzymes and ligase. The resulting clones maintain the original orientation and reading frame allowing functional analysis of genes and whole libraries.
Why a Full-Length cDNA Library Construction Kit?
The SuperScript® Full-Length cDNA Library Construction Kit allows you to: • Prepare next-generation sequencing templates from full-length-selected transcripts • Identify exact transcription start sites • Discover different isoforms or alternatively spliced genes that are expressed in different cells⁄tissues • Discover sequence motifs located near the 5' end of the gene • Obtain full-length cDNA sequences to enable identification of gene families, and thus discover new family members without further labor-intensive efforts such as 5' RACE.
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