[Protocol] Extraction of chromosomal DNA from Schizosaccharomyces pombe

Murray, Johanne, Watson, Adam and Carr, Antony (2016) [Protocol] Extraction of chromosomal DNA from Schizosaccharomyces pombe. Cold Spring Harbor Protocols, 2016 (5). ISSN 1559-6095

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Abstract

Extraction of DNA from Schizosaccharomyces pombe cells is required for various uses, including templating polymerase chain reactions (PCRs), Southern blotting, library construction, and high-throughput sequencing. To purify high-quality DNA, the cell wall is removed by digestion with Zymolyase or Lyticase and the resulting spheroplasts lysed using sodium dodecyl sulfate (SDS). Cell debris, SDS, and SDS–protein complexes are subsequently precipitated by the addition of potassium acetate and removed by centrifugation. Finally, DNA is precipitated using isopropanol. At this stage, purity is usually sufficient for PCR. However, for more sensitive procedures, such as restriction enzyme digestion, additional purification steps, including proteinase K digestion and phenol–chloroform extraction, are recommended. All of these steps are described in detail here.

Item Type: Article
Schools and Departments: School of Life Sciences > Sussex Centre for Genome Damage and Stability
Research Centres and Groups: Genome Damage and Stability Centre
Depositing User: Sarah Frances
Date Deposited: 13 Dec 2017 14:03
Last Modified: 22 Jan 2018 13:28
URI: http://sro.sussex.ac.uk/id/eprint/72008

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