The Nav channel bench series: Plasmid preparation.
The Nav channel bench series: Plasmid preparation.
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DOI：
10.1016/j.mex.2014.01.002
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发表时间：
2014
期刊：
影响因子：
1.9
通讯作者：
Lossin, Christoph
中科院分区：
文献类型：
--
作者：
Feldman, Daniel H;Lossin, Christoph
使用Stbl2细胞在30°C或以下进行细菌繁殖。偏爱生长缓慢的小菌落。全面排序整个导航频道编码区域。涉及重组电压门控钠（Nav）通道的研究具有独特的挑战。多种因素都有影响，但毫无疑问，最重要的因素是这些通道的DNA不稳定性。一旦引入细菌宿主，除非采用特殊条件，否则Nav通道质粒DNA几乎总是会发生突变而无法使用。对于Nav1.1（基因名称SCN1A）、Nav1.2 （SCN2A）和Nav1.6 （SCN8A）来说尤其如此，但对于Nav1.4 （SCN4A）和Nav1.5 （SCN5A）来说就不那么明显了，而其他的nav通道亚型如Nav1.7 （SCN9A）则介于两者之间。以下关于Nav质粒DNA扩增和制备的建议解决了这个问题。有三点很重要：
Bacterial propagation using Stbl2 cells at or below 30 °C. Bias toward slow-growing, small bacterial colonies. Comprehensive sequencing of the entire Nav channel coding region. Research involving recombinant voltage-gated sodium (Nav) channels has unique challenges. Multiple factors contribute, but undoubtedly at the top of the list is these channels’ DNA instability. Once introduced into bacterial hosts, Nav channel plasmid DNA will almost invariably emerge mutagenized and unusable, unless special conditions are adopted. This is particularly true for Nav1.1 (gene name SCN1A), Nav1.2 (SCN2A), and Nav1.6 (SCN8A), but less so for Nav1.4 (SCN4A) and Nav1.5 (SCN5A) while other Nav channel isoforms such as Nav1.7 (SCN9A) lie in between. The following recommendations for Nav plasmid DNA amplification and preparation address this problem. Three points are essential: