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DNA Isolation

DNA Isolation

DNA isolation protocols can be fast and straightforward or complex and time-consuming depending on the type and quality of DNA required for further processing. For example, plasmid DNA extraction is a bit more complicated than genomic DNA extraction because of the need for an appropriate lysis method to separate plasmid DNA from gDNA during isolation. However, for specific applications, such as long-range DNA sequencing that require a good yield of high- quality DNA samples, we need to follow...

PCR

PCR

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Related Experiment Video

Updated: Dec 6, 2025

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

Published on: March 10, 2020

Cloning short DNA into plasmids by one-step PCR.

Cheng-Cheng Tao¹, Ying Yang¹, Fang Li²

¹West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, Chengdu, China.

Thoracic Cancer

|October 5, 2020

Summary

A novel one-step Polymerase Chain Reaction (PCR) cloning method enables efficient insertion of short DNA fragments into plasmids. This technique bypasses traditional restriction enzyme limitations, offering a flexible and economical approach for molecular biology research.

Keywords:

Homologous recombination one-step PCR plasmid construction restriction enzyme-based cloning method

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Last Updated: Dec 6, 2025

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Area of Science:

Molecular Biology
Biotechnology

Background:

Plasmid construction is fundamental for biomolecular experiments, including marker gene insertion and gene expression modulation.
Traditional methods often rely on restriction enzymes and ligation, which can be limiting.

Purpose of the Study:

To introduce a streamlined method for cloning short DNA fragments into plasmids.
To provide a flexible and economical alternative to existing plasmid construction techniques.

Main Methods:

A one-step Polymerase Chain Reaction (PCR) cloning approach was developed.
Primers with 10-15 bp overlapping sequences amplified the target DNA and the entire circular plasmid.
PCR products were transformed into E. coli for in vivo homologous recombination and cyclization.

Main Results:

Successfully constructed the pEGFP-N1-HA plasmid using the one-step PCR cloning method.
Confirmed HA expression in cells transfected with the constructed plasmid.
Detected HA-GFP fusion protein using western blotting and confocal microscopy.

Conclusions:

The one-step PCR cloning method eliminates the need for restriction enzyme sites and ligation.
This technique offers a flexible, economical, and efficient option for plasmid construction.
It simplifies the process of inserting short DNA fragments into vectors.