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一种从棉花(棉属)中快速提取DNA的改良方法。

A modified protocol for rapid DNA isolation from cotton ( spp.).

作者信息

Ali Qasim, Salisu Ibrahim Bala, Raza Ali, Shahid Ahmad Ali, Rao Abdul Qayyum, Husnain Tayyab

机构信息

Centre of Excellence in Molecular Biology, University of the Punjab, Lahore-54000, Pakistan.

Department of Animal Science, Faculty of Agriculture, Federal University Dutse, P.M.B. 7156, Jigawa State, Nigeria.

出版信息

MethodsX. 2019 Jan 30;6:259-264. doi: 10.1016/j.mex.2019.01.010. eCollection 2019.

Abstract

Extraction of high-quality DNA from (Cotton) species is notoriously difficult due to high contents of polysaccharides, quinones and polyphenols other secondary metabolites. Here, we describe a simple, rapid and modified procedure for high-quality DNA extraction from cotton, which is amenable for downstream analyses. In contrast to other CTAB methods, the described procedure is rapid, omits the use of liquid nitrogen, phenol, CsCl gradient ultracentrifugation, uses inexpensive and less hazardous reagents, and requires only ordinary laboratory equipment. The procedure employed the high concentration of NaCl and use of PVP-10 to rid the problems associated with polysaccharides and polyphenols, respectively. The average yield was approximately 10-15 μg of good quality DNA from 100 mg of tissue weight, which is adequate for projects, like genetic mapping or marker-assisted plant breeding. This protocol can be performed in as little as 3 h and may be adapted to high-throughput DNA isolation. •Buffers A and B were redesigned from Paterson et al. (1993) and Porebski et al. (1997), respectively.•Ribonuclease A was added before chloroform extraction.•A simple, rapid and inexpensive DNA extraction method is described.

摘要

从棉花物种中提取高质量的DNA非常困难,因为其含有大量的多糖、醌类和多酚等次生代谢产物。在此,我们描述了一种简单、快速且经过改进的从棉花中提取高质量DNA的方法,该方法适用于下游分析。与其他十六烷基三甲基溴化铵(CTAB)方法不同,所描述的方法快速,无需使用液氮、苯酚、氯化铯梯度超速离心,使用的试剂价格低廉且危害较小,仅需普通实验室设备。该方法采用高浓度氯化钠和使用聚乙烯吡咯烷酮-10(PVP-10)分别解决与多糖和多酚相关的问题。平均产量约为每100毫克组织重量产生10 - 15微克高质量DNA,这对于基因图谱绘制或标记辅助植物育种等项目来说足够了。该方案最短可在3小时内完成,并且可适用于高通量DNA分离。•缓冲液A和B分别根据帕特森等人(1993年)和波雷布斯基等人(1997年)的方法重新设计。•在氯仿提取前添加核糖核酸酶A。•描述了一种简单、快速且廉价的DNA提取方法。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9b55/6370549/b247cd344383/fx1.jpg

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