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The Dynabeads® mRNA DIRECT™ Kit is designed for simple and rapid isolation of pure, intact polyadenylated (polyA) mRNA directly from the crude lysate of animal and plant cells and tissues. The isolated mRNA is suitable for use in all downstream applications. Advantages of the Dynabeads® mRNA DIRECT™ Kit: • Fast—15 minute procedure yields pure, intact mRNA • Highly pure mRNA isolation—best choice upstream of cDNA synthesis • Sensitive mRNA isolation—enables cDNA synthesis and cDNA library construction from ultra-small starting samples (enables cDNA library construction from a single cell) System overview The isolation protocol relies on base pairing between the polyA residues at the 3' end of most mRNA, and the oligo (dT)25 residues covalently coupled to the surface of the Dynabeads® . Other RNA species lacking a polyA tail will not hybridize to the beads and are readily washed away. Ribosomal RNA, DNA, proteins, and small RNA molecules (such as transfer RNA, micro RNA, and small nucleolar RNA) do not bind to the beads and are discarded. RNase inhibiting agents in the Lysis/Binding Buffer together with stringent hybridization and washing conditions ensure the isolation of pure, intact mRNA from crude samples rich in RNase, without the use of strong chaotropic agents. The mRNA purification beads specifically target and capture the mRNA transcriptome from an extremely wide variety of crude starting samples (see protocol). 1 mg of Dynabeads® Oligo (dT)25 beads (200 µL) binds up to 2 µg of mRNA. A typical mammalian cell contains about 10–30 pg of total RNA, from which 1–5% is mRNA. |
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