Isolation of high-quality DNA from cotton and its fungal pathogens
Isolation of high-quality DNA from cotton and its fungal pathogens
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DOI：
10.1007/bf03356717
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发表时间：
2007-06-01
影响因子：
2
通讯作者：
El-Samawaty, A. M. A.
中科院分区：
文献类型：
--
作者：
Abd-Elsalam, K. A.;Asran-Amal, A.;El-Samawaty, A. M. A.
为了从棉花不同组织(叶片和种子)以及菌丝体中提取高质量的复杂基因组DNA，建立了一种单种子DNA提取方法。它的真菌病原体。种子在24孔细胞培养群中萌发，取5日龄的幼苗组织提取DNA。提取的DNA的平均产量从每100毫克处理的组织中获得50到100毫克的DNA，允许超过150次的PCR实验。为了考察本方法的有效性，对DNA提取液进行了不同的处理，包括限制性内切酶酶切、随机扩增和ITS-PCR扩增。在所有情况下，在不到60min的时间内就可以制备出适合于PCR和限制性内切酶的DNA。本文描述的方法可以很好地从棉花组织中提取高质量的DNA，并应广泛适用于所有棉花基因型及其真菌病原体的大群体分析。该方法不需要昂贵且对环境有害的试剂和设备。即使在低技术的实验室里也可以进行。
A single-seed DNA extraction method was developed to extract high quality complex genomic DNA from different cotton tissues (leaves and seeds) as well as from mycelium. of its fungal pathogens. Seeds were germinated in 24-well cell culture cluster and 5-days-old seedling tissue was used to extract DNA. The average yield of extracted DNA varied from 50 to 100 ng of DNA per 100 mg of processed tissue, allowing over 150 PCR experiments. To investigate the effectiveness of our protocol, DNA extracts were subjected to different treatments, including restriction enzyme digestion, RAPD and ITS-PCR amplification. in all cases, DNA suitable for PCR and for restriction was prepared in less than 60 min. The procedure described here works well for extracting high-quality DNA from cotton tissue and should be widely applicable for an analysis of large populations from all cotton genotypes and its fungal pathogens. The method does not require expensive and environmentally hazardous reagents and equipment. It can be performed even in low-technology laboratories.