从不同的反抗性细菌物种中分离高质量的等离子体的协议:Agrobacterium spp,Rhizobium sp.和Bacillus thuringiensis

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Preshobha Kodackattumannil¹, Shina Sasi¹, Saranya Krishnan¹

¹Khalifa Center for Genetic Engineering and Biotechnology, United Arab Emirates University, P.O. Box 15551, Al Ain, United Arab Emirates.

Bio-protocol

|August 14, 2023

概括

这项研究介绍了一种修改后的等离子体提取协议,可以有效地将高质量的等离子体DNA从具有挑战性的细菌物种如Agrobacterium和Bacillus thuringiensis中分离出来. 优化的方法避免了提取和密度梯度,产生下游应用的测序级DNA.

科学领域:

分子生物学分子生物学
微生物学微生物学
生物技术是生物技术.

背景情况:

标准的等离子体DNA提取套件通常会产生少量的质量差的等离子体DNA,这些DNA来自格兰阴性细菌 (如Agrobacterium,Rhizobium) 和格兰阳性细菌 (如Bacillus thuringiensis).
高质量的等离子体DNA的有效隔离对于各种分子生物学应用至关重要,包括基因工程和转基因组学研究.

研究的目的:

开发和优化难以溶解的细菌物种的内部等离子体DNA提取协议.
为了提高等离子体DNA的产量和质量,用于下游应用,包括直接转换和测序.

主要方法:

修改了Qiagen QIAprep Spin Miniprep套件协议,其中包括两个提取步骤.
在中和和化后加入乙醇沉和化:异胺醇 (CIA) 提取.
包括RNase处理和最终沉和洗步骤来净化等离子体DNA.

主要成果:

优化的协议成功地产生了大量来自Agrobacterium tumefaciens,A. rhizogenes,Rhizobium sp.和Bacillus thuringiensis的优质等离子体DNA.
提取的等离子体DNA是测序级的,适用于高通量下游应用.
该协议不含,避免密度梯度步骤,不需要DNA结合列.

结论:

这种修改后的等离子体提取协议提供了一种高效,易于执行和可扩展的方法,用于从具有挑战性的细菌物种中分离高质量的等离子体DNA.
该协议促进了直接转化为Agrobacterium,绕过了Escherichia coli转化的需要.
该方法对于诸如元基因组研究和高通量遗传分析等应用非常有价值.

关键词:

农业细菌 (Agrobacterium) 是一种细菌.细菌 thuringiensis 这种疾病.低复制数细菌菌株的细菌菌株.修改后的QIAprep Spin Miniprep套件协议修改后的QIAprep套件协议塑体DNA DNA 的意思.化是一种化.硫酸二甲二氧化硫酸是什么

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