Protocol

Bidirectional Cloning of PCR Products

This protocol was adapted from "Bidirectional and Directional Cloning of PCR Products," contributed by Gina L. Costa and Michael P. Weiner, Chapter 28, in PCR Primer, 2nd edition (eds. Dieffenbach and Dveksler). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003.

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INTRODUCTION

This protocol is for bidirectional, blunt-end cloning of DNA fragments. The target DNA is PCR amplified and 3'-extensions are polished with Pfu DNA polymerase. The amplicon is ligated to a blunt-ended plasmid DNA, and the products of the ligation reaction are used to transform competent Escherichia coli. A restriction enzyme is added to the ligation reaction (Liu and Schwartz 1992) to relinearize any self-religating vector DNA.

MATERIALS

Reagents

Ammonium acetate, 4 M (optional, see Step 11)

ATP, 10 mM

Betaine solution, 5 M, PCR-grade (Sigma)

Blunt-end restriction endonucleases, e.g., SrfI and SmaI (10-20 units)

Phenol:chloroform:isoamylalcohol (25:24:1, v/v)

Cloning vector DNA

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Equipment

METHOD