Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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In Vitro Mutagenesis Using Double-Stranded DNA Templates: Selection of Mutants with DpnI

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In both this protocol and Protocol 5, two oligonucleotides are used to prime DNA synthesis by a high-fidelity polymerase on a denatured plasmid template. The two oligonucleotides both contain the desired mutation and have the same starting and ending positions on opposite strands of the plasmid DNA. In this protocol, the entire lengths of both strands of the plasmid DNA are amplified in a linear fashion during several rounds of thermal cycling, generating a mutated plasmid containing staggered nicks on opposite strands (see Fig. 1) (Hemsley et al. 1989).


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(Limited time special offer.) Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook
Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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