An optimized protocol for the creation of constructs expressing sodium channel subunits to be used in functional studies of genetic epilepsies

Abstract

ABSTRACTBackgroundWe aim to develop a method for cloning highly unstable plasmids encoding sodium channel subunits expressed in the brain.MethodNEB Stable competent cells (NSCC) were transformed with the native plasmid. After purification, plasmids with the expected profile of enzymatic digestion were sequenced. Finally, mutant linear plasmids (MLP) created by site-directed mutagenesis were used to transform NSCC.ResultsWe found that choosing a suitable host for transformation and subcloning, the controlled temperature during cell growth, and a small volume of Luria Bertani (LB) medium allowed the generation of plasmids without undesirable mutations or structural alterations.Comparison with existing methodAt present, there is little information on a detailed method for cloning plasmids carrying the SCN1A gene. Using the method proposed here, we observed increased cell growth and precision in the sequence of the obtained plasmids.ConclusionWe have developed an optimized strategy for replicating unstable plasmids using NSCC with an appropriate volume of culture medium and an adequate temperature in the cloning and subcloning phases. We have also demonstrated how to create the desired plasmid constructs by using site-directed mutagenesis. We expect that our protocol will assist investigators interested in functional studies of sodium channels expressed in the brain.