Isolation of dna and rna from Streptococcus sobrinus OMZ176 using CsTFA gradients |
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Affiliation: | 1. Centre for Infection and Immunity, Queens University Belfast, Northern Ireland, United Kingdom;2. Regional Virus Laboratory/Department of Microbiology, Belfast Health and Social Care Trust, Northern Ireland, United Kingdom;3. Royal Belfast Hospital for Sick Children, Northern Ireland, United Kingdom |
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Abstract: | - 1.1. A simple procedure for isolation of high molecular weight genomic DNA, and RNA, from Streptococcus sobrinus OMZ176 is described.
- 2.2. Cell cultures were grown aerobically for 10 hr.
- 3.3. Spheroplast formation and lysis was achieved by mutanolysin/lysozyme-dependent digestion of the cell wall, followed by N-lauroylsarcosinate-mediated lysis.
- 4.4. Nucleic acids were isolated directly from cell-lysates using cesium-trifluoroacetate (CsTFA) densitygradient centrifugation.
- 5.5. Three different centrifugation regimes were tested: self-generated density gradients in a fixed angle rotor; self-generated density-gradients in a swinging-bucket rotor; pre-formed density-gradients in a swinging-bucket rotor.
- 6.6. Genomic DNA isolated by the CsTFA-procedure was found to have higher purity as compared to genomic DNA isolated in a conventional CsCl gradient.
- 7.7. Isolated DNA was shown to be of a quality suitable for applications in molecular biology.
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