“First Generation” Automated DNA Sequencing Technology
New England Biolabs, Inc., Ipswich, Massachusetts, USA.Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.] 10/2011; Chapter 7:Unit7.2. DOI: 10.1002/0471142727.mb0702s96
Beginning in the 1980s, automation of DNA sequencing has greatly increased throughput, reduced costs, and enabled large projects to be completed more easily. The development of automation technology paralleled the development of other aspects of DNA sequencing: better enzymes and chemistry, separation and imaging technology, sequencing protocols, robotics, and computational advancements (including base-calling algorithms with quality scores, database developments, and sequence analysis programs). Despite the emergence of high-throughput sequencing platforms, automated Sanger sequencing technology remains useful for many applications. This unit provides background and a description of the "First-Generation" automated DNA sequencing technology. It also includes protocols for using the current Applied Biosystems (ABI) automated DNA sequencing machines.
[Show abstract] [Hide abstract]
- "tissue and smaller cells. 28. Carefully layer the 1 ml of cell suspension on top of the Percoll/L15 medium mixture prepared in step 23 by pipetting the cell suspension slowly and steadily onto the wall of the tube close to the Percoll/L15 mixture, holding the tube at a 60 @BULLET angle. Gently move the tube back to a vertical position and close it.Slatko et al., 2011)."
ABSTRACT: Airway sensory nerves play an important defensive role in the lungs, being central in mediating protective responses like cough and bronchoconstriction. In some cases, these responses become excessive, hypersensitive, and deleterious. Understanding the normal function of airway nerves and phenotype changes associated with disease will help in developing new therapeutics for treating chronic obstructive pulmonary disease and chronic cough. Guinea pigs, and to a lesser extent ferrets, are commonly employed for studying the cough reflex because they have a cough response similar to humans. While rats and mice do not exhibit a cough response, they do possess sensory nerves that respond to the same range of tussive stimuli as guinea pigs and humans. Described in this unit are protocols for harvesting guinea pig, mouse, and rat sensory nerve cell bodies to assess molecular and functional changes associated with pulmonary disease, and to identify new targets for therapeutic intervention. Curr. Protoc. Pharmacol. 62:12.15.1-12.15.27. © 2013 by John Wiley & Sons, Inc.