Protocol

Ribocloning: DNA Cloning and Gene Construction Using PCR Primers Terminated with a Ribonucleotide

This protocol was adapted from "Ribocloning: DNA Cloning and Gene Construction Using PCR Primers Terminated with a Ribonucleotide," contributed by Wayne M. Barnes, Chapter 29, 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 exploits the discovery that RNase A can efficiently cleave at single rC or rU bases embedded in double-stranded DNA. Entire plasmid vectors are amplified using long, high-fidelity PCR with riboprimers, which carry a single rC residue at their 3' end. Target DNA is amplified using similar primers, which also end in a rC residue. Both target and vector DNA are then subjected to RNase A treatment, which introduces a nick at the rC site, between the primer and newly synthesized DNA. Heating the nicked molecules causes the primer sequences to "peel off," leaving extended "sticky" ends on the . . . [Full Text of this Article]

MATERIALS

Reagents

Equipment

METHOD

TROUBLESHOOTING