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In-vitro Mutagenesis

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Related Experiment Video

Updated: Aug 8, 2025

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Published on: February 5, 2019

In Vitro Site Directed Mutagenesis.

Michael J McClellan¹

¹Ludwig Institute for Cancer Research Ltd, University of Oxford, Oxford, UK. michael.mcclellan@ludwig.ox.ac.uk.

Methods in Molecular Biology (Clifton, N.J.)

|February 28, 2023

Summary

This summary is machine-generated.

Site-Directed Mutagenesis (SDM) enables precise DNA modifications in plasmids. This study details a rapid, ligation-free SDM protocol for genetic experiments, followed by colony PCR screening.

Keywords:

Colony PCR Genetic alteration Mutation Plasmid manipulation Polymerase chain reaction Site-directed mutagenesis

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Last Updated: Aug 8, 2025

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

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Published on: February 5, 2019

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Published on: May 11, 2009

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity

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Published on: March 25, 2020

Area of Science:

Molecular Biology
Genetic Engineering
Biochemistry

Background:

Site-Directed Mutagenesis (SDM) is a fundamental technique for altering DNA sequences.
It serves as a crucial initial step in numerous biochemical and genetic research.
Existing SDM methods can be time-consuming or require multiple steps.

Purpose of the Study:

To describe various forms of Site-Directed Mutagenesis.
To present a detailed, fast, and ligation-free protocol for SDM.
To enable efficient screening of mutated sequences using colony PCR.

Main Methods:

Utilized polymerase chain reaction (PCR) for DNA sequence modification.
Developed and implemented a ligation-free protocol for introducing substitutions, insertions, or deletions.
Employed colony PCR for rapid screening of desired mutations.

Main Results:

Successfully demonstrated a streamlined SDM approach.
The ligation-free protocol proved efficient for introducing various DNA alterations.
Colony PCR effectively identified mutated sequences, confirming the protocol's utility.

Conclusions:

The described ligation-free SDM protocol is a reliable, rapid, and accessible method.
This technique simplifies the initial steps of many molecular biology experiments.
It offers an efficient alternative for generating DNA sequence modifications.