Protocol

Construction of cDNA Libraries

Stage 2: Second-strand Synthesis

This protocol was adapted from Molecular Cloning, 3rd edition, by Joseph Sambrook and David W. Russell. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2001

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INTRODUCTION

Here, the DNA-RNA hybrids synthesized in Stage 1 are converted into full-length double-stranded cDNAs. The primers for synthesis of second-strand cDNA are created by RNase H, which introduces nicks into the RNA moiety of the cDNA-mRNA hybrids. E. coli DNA polymerase I extends the newly created 3'-hydroxyl termini, using the first-strand cDNA as a template. In this way, the remaining segments of mRNA in the cDNA-mRNA hybrid are replaced with the newly synthesized second strand of DNA. Residual nicks are then repaired by E. coli DNA ligase, and the frayed termini of the double-stranded cDNA are polished by a DNA . . . [Full Text of this Article]

MATERIALS

METHOD