Protocol

Construction and Screening of Eukaryotic Expression Libraries

Stage 1: Construction of cDNA Libraries in Eukaryotic Expression Vectors

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

This stage summarizes how to construct a cDNA library in a mammalian vector, using commercial kits. These kits are available from several manufacturers and generally include all the required reagents.

MATERIALS

10x Universal KGB buffer (restriction endonuclease buffer[s])

Electrocompetent E. coli cells

If a plasmid expression vector is chosen, prepare (please see Transformation of E. coli by Electroporation) or purchase electrocompetent E. coli as hosts for the cDNA library. The titer of the electrocompetent cells should be at least 5 x 10⁸ colonies/µg plasmid DNA. Ideally, use a single lot or preparation of cells throughout the expression cloning and screening . . . [Full Text of this Article]