Isolation of Genomic DNA from Mycobacterium Species
从分枝杆菌物种中分离基因组DNA
(*contributed equally to this work) 发布: 2016年03月05日第6卷第5期 DOI: 10.21769/BioProtoc.1751 浏览次数: 16415
评审: Ivan ZanoniRamalingam BethunaickanBenoit Chassaing
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参见作者原研究论文
The authors used this protocol in:
Mar 2015
Abstract
Isolation of genomic DNA from Mycobacterium species has been a tedious procedure. This can primarily be attributed to thick and waxy cell wall of mycobacteria which hampers lysis of the bacterial cell. We have tested various approaches to isolate mycobacterial DNA and based on this, an optimized protocol is presented here. This protocol involves initial incubation of mycobacteria with lysozyme, followed by SDS-proteinase K treatment to bring about cell disruption. In the case of slowly growing mycobacteria such as BCG (Bacillus Calmette–Guérin) or Mycobacterium tuberculosis (M. tuberculosis), an intermediate step of cell lysis by physical method results in significantly enhanced yield.
Materials and Reagents
- Mycobacterium culture (50 to 100 ml)
- Tris base (Sigma-Aldrich, catalog number: T1503 )
- EDTA (HiMedia Laboratories, catalog number: MB011 )
Note: Prepare 0.5 M solution (pH 8) in double distilled water.
- Liquid nitrogen
- Proteinase K (HiMedia Laboratories, catalog number: RM2957 )
Note: Prepare the stock solution of 10 mg/ml in TE buffer (pH 8).
- SDS (Bio Basic Canada, catalog number: SB0485 )
Note: Prepare 10% solution by dissolving in double distilled water.
- Lysozyme (Sigma-Aldrich, catalog number: L6876 )
Note: Prepare the stock solution of 100 mg/ml in double distilled water.
- Phenol (Sigma-Aldrich, catalog number: P4557 )
Note: Equilibrate with Tris-Hcl (pH 8).
- Chloroform (HiMedia Laboratories, catalog number: AS041 )
- Isoamyl alcohol (HiMedia Laboratories, catalog number: MB091 )
- 2-Propanol (HiMedia Laboratories, catalog number: MB063 )
- Ethyl alcohol, Pure (Sigma-Aldrich, catalog number: 459844 )
- Nuclease-free water (HiMedia Laboratories, catalog number: TCL016 )
- TE buffer (see Recipes)
- PCI mixture (see Recipes)
Equipment
- Water bath
- Centrifuge
- Pestle and mortar
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Kumar, P., Marathe, S. and Bhaskar, S. (2016). Isolation of Genomic DNA from Mycobacterium Species. Bio-protocol 6(5): e1751. DOI: 10.21769/BioProtoc.1751.
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分类
微生物学 > 微生物遗传学 > DNA
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