相关实验视频

Updated: Jun 6, 2025

Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Published on: January 8, 2015

RecombiCraft图书馆建设:一种新的DNA图书馆克隆和扩展方法,使用非酶的单步DNA重组和液体培养

Yuki Kawai-Harada^1,2, Mehrsa Mardikoraem^1,3, Katherine Lauro^1,2

¹Institute for Quantitative Health Science and Engineering, Division of Chemical Biology, Michigan State University, East Lansing, Michigan, United States of America.

|December 2, 2024

概括

在大肠杆菌中,RecombiCraft使用无联结克隆提取物 (SLiCE) 和液体培养物快速构建DNA库. 这种成本效益高的方法最大限度地减少了序列偏差,为遗传研究提供了一种简化方法.

科学领域:

分子生物学分子生物学
合成生物学合成生物学
生物技术是生物技术.

背景情况:

高效的DNA库建设对于遗传研究和生物技术至关重要.
现有的方法可能是耗时的,昂贵的,容易产生序列偏差.
需要快速,经济和最小化偏差的DNA库生成技术.

研究的目的:

引入RecombiCraft,一种用于在大肠杆菌中构建DNA库的新方法.
为了证明RecombiCraft的速度,成本效益和最小的序列偏差.
为基因工程研究人员提供简化和可访问的工具.

主要方法:

使用无结合克隆提取物 (SLiCE) 进行DNA片段组装.
在大肠杆菌中使用液体培养放大,以利用自然同源重组.
通过PCR与高保真聚合酶合成图书馆的骨干和插件.
优化培养时间,使用下一代测序进行偏差评估.

主要成果:

RecombiCraft成功地生成了具有最小序列偏差的DNA库.
这种方法被证明是具有成本效益的,因为它减少了对多个纯化的酶的需求.
在大约12小时内完成了DNA库的生成.
液体培养放大和简单的提取有助于该方法的效率.

结论:

RecombiCraft提供了一个创新的,快速和经济的解决方案,用于DNA库的构建.
该方法保持了库的统一性,并有效地减少了序列偏差.
RecombiCraft是推动遗传研究和生物技术应用的一个有前途的工具.

更多相关视频

Recombineering Homologous Recombination Constructs in Drosophila

Recombineering Homologous Recombination Constructs in Drosophila

Published on: July 13, 2013

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Published on: December 1, 2017

相关概念视频

Recombinant DNA

Recombinant DNA

Conservative Site-specific Recombination and Phase Variation

Conservative Site-specific Recombination and Phase Variation

Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...

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...

Homologous Recombination

Homologous Recombination

The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...