Resource Summary Report  New Search Previous Search Results

URL: https://dx.doi.org/DOI:10.17504/protocols.io.bf28jqhw

Authors: Leah Cuthbertson, Michael Cox, Phillip James , Colin Churchward, Bill Cookson, Miriam Moffatt

Summary: This extraction method is modified from Griffiths et al 2000 Applied Environmental Microbiology 66(12): 5488–5491 and DeAngelis et al 2009 ISME Journal 3 pp 168-178.In comparison with DNA extraction kits, nucleic acid yields tend to be higher, though it is more laborious for larger numbers of samples.The extraction uses CTAB and Phenol:Chloroform:Isoamyl alcohol for lysis and the precipitation of nucleic acid is PEG based. An alternative to bead-beating is described, which should only be used for extracting DNA from cultured isolates. Bead-beating is avoided in this case in order to minimise shearing of DNA prior to genomic DNA sequencing.For all other samples use bead-beating is recommended.This Protocol will cover the extraction of the following sample types;Bacterial isolatesUpper respiratory tract swabs (nasal and throat swabs)Upper respiratory tract lavage and aspirates (nasopharyngeal aspirate, endotracheal aspirate, nasal lavage, Mouth wash)SalivaSputum (expectorated spontaneously produced, and induced sputum)Upper and lower respiratory tract synthetic absorptive matrix (SAM) stripsLower respiratory tract samples obtained via bronchoscopy - lung brushes, bronchoalveolar lavage and pleural fluidLung tissue – human or mouseLung biopsiesCerebral spinal fluid (CSF)This is a double extraction protocol, you are adding a second volume of Phenol:Chloroform:Isoamyl alcohol to each sample, precipitating two aqueous phases per sample and then recombining the resulting DNA pellets.This approach significantly increases the yield of DNA from samples and strains.

Affiliations: National Heart and Lung Institute, Imperial College, London, UK, SW3 6LY, National Heart and Lung Institute, Imperial College, London, UK, SW3 6LY, National Heart and Lung Institute, Imperial College, London, UK, SW3 6LY, National Heart and Lung Institute, Imperial College, London, UK, SW3 6LY, National Heart and Lung Institute, Imperial College, London, UK, SW3 6LY, National Heart and Lung Institute, Imperial College, London, UK, SW3 6LY

Version: 1

Publication Date: 2020

Expand All Collapse