The Nav channel bench series: Plasmid preparation

MethodsX. 2014 Feb 1;1:6-11. doi: 10.1016/j.mex.2014.01.002. eCollection 2014.

Abstract

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:•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.

Keywords: Channelopathy; DNA; Mutation; Nav channel; Nav1.1; Nav1.6; Plasmid; SCN1A; SCN8A; Sodium channel; Stbl2.