The Nav channel bench series: Plasmid preparation
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 an...
| Main Authors: | , |
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| Format: | Article |
| Language: | English |
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Elsevier
2014-01-01
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| Series: | MethodsX |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S221501611400003X |
| _version_ | 1828422396229451776 |
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| author | Daniel H. Feldman Christoph Lossin |
| author_facet | Daniel H. Feldman Christoph Lossin |
| author_sort | Daniel H. Feldman |
| collection | DOAJ |
| description | 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:
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Bacterial propagation using Stbl2 cells at or below 30 °C.
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Bias toward slow-growing, small bacterial colonies.
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Comprehensive sequencing of the entire Nav channel coding region. |
| first_indexed | 2024-12-10T15:46:24Z |
| format | Article |
| id | doaj.art-270c57799f754bb4830dcec447c1a793 |
| institution | Directory Open Access Journal |
| issn | 2215-0161 |
| language | English |
| last_indexed | 2024-12-10T15:46:24Z |
| publishDate | 2014-01-01 |
| publisher | Elsevier |
| record_format | Article |
| series | MethodsX |
| spelling | doaj.art-270c57799f754bb4830dcec447c1a7932022-12-22T01:42:56ZengElsevierMethodsX2215-01612014-01-011C61110.1016/j.mex.2014.01.002The Nav channel bench series: Plasmid preparationDaniel H. FeldmanChristoph LossinResearch 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.http://www.sciencedirect.com/science/article/pii/S221501611400003XSodium channelNav channelMutationPlasmidDNANav1.1SCN1ANav1.6SCN8AStbl2Channelopathy |
| spellingShingle | Daniel H. Feldman Christoph Lossin The Nav channel bench series: Plasmid preparation MethodsX Sodium channel Nav channel Mutation Plasmid DNA Nav1.1 SCN1A Nav1.6 SCN8A Stbl2 Channelopathy |
| title | The Nav channel bench series: Plasmid preparation |
| title_full | The Nav channel bench series: Plasmid preparation |
| title_fullStr | The Nav channel bench series: Plasmid preparation |
| title_full_unstemmed | The Nav channel bench series: Plasmid preparation |
| title_short | The Nav channel bench series: Plasmid preparation |
| title_sort | nav channel bench series plasmid preparation |
| topic | Sodium channel Nav channel Mutation Plasmid DNA Nav1.1 SCN1A Nav1.6 SCN8A Stbl2 Channelopathy |
| url | http://www.sciencedirect.com/science/article/pii/S221501611400003X |
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