Abstract
This chapter discusses the generation of cDNA insert from eukaryotic messenger RNA (mRNA). mRNA is isolated from eukaryotic cells and generates specific cDNA for insertion into λgt10 or λgt11 phage. This is a long, extensive procedure for preparing an insert. A scintillation counter is required for this purpose. Total RNA from tissue of interest is prepared and poly (A + ) RNA is selected. dT-selected RNA is ready for cDNA preparation and library construction. The sample is stored at 1 μg poly (A + ) RNA per microliter of H 2 O at -70°C. The chapter explains step by step the preparation of cDNA, Eco RI methylation reaction, Eco RI linker addition, Eco RI digestion to generate cohesive Eco RI cloning ends, and size fractionation of cDNA insert.
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