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Related Experiment Video

Updated: Jul 4, 2026

CRISPR-based Shuttle Cloning: A High-throughput Cloning Method

CRISPR-based Shuttle Cloning: A High-throughput Cloning Method

Published on: June 13, 2025

High throughput cloning with restriction enzymes.

Volker Sievert¹, Asgar Ergin, Konrad Büssow

¹Max Planck Institute for Molecular Genetics, Department of Vertebrate Genomics, Protein Structure Factory, Berlin, Germany.

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

|June 11, 2008

Summary

High-throughput cloning of bacterial expression clones for human proteins is essential for structural genomics. This study details a 96-well plate method using specific enzymes for efficient protein structure analysis.

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Area of Science:

Structural biology
Genomics
Molecular biology

Background:

Systematic structural analysis of proteins requires high-throughput generation of expression clones.
Laboratory automation and data management are crucial for efficient cloning processes.
Structural genomics projects focus on understanding protein structures on a large scale.

Purpose of the Study:

To present an optimized method for cloning bacterial expression constructs for human proteins.
To adapt cloning procedures to a 96-well microtiter plate format for high-throughput applications.
To provide an overview of the Protein Structure Factory's approach to structural genomics.

Main Methods:

Utilized restriction enzymes BamHI and NotI, along with compatible enzymes, for cloning.
Adapted Polymerase Chain Reaction (PCR) amplification for high-throughput clone generation.
Implemented purification, digestion, and vector ligation steps in a 96-well microtiter plate format.

Main Results:

Successfully adapted key molecular cloning steps (PCR, purification, digestion, ligation) to a 96-well format.
Developed a robust procedure for generating bacterial expression clones suitable for structural analysis.
Demonstrated the feasibility of high-throughput cloning within the Protein Structure Factory project.

Conclusions:

The described 96-well plate method enables efficient, high-throughput cloning of bacterial expression constructs.
This approach supports large-scale structural genomics efforts, particularly for human proteins.
Automation and standardized protocols are key to advancing structural biology research.