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  • Genomic DNA Isolation From Bacteria

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    This document provides a protocol for isolating genomic DNA from bacteria using triton purification. The key steps are: 1) Grow bacterial colonies in large-scale cultures and harvest the cells into a tube. 2) Resuspend the pellet in STET buffer and add a lysozyme/RNase mixture. Boil the mixture for 1 minute and 15 seconds. 3) Centrifuge the mixture and collect the supernatant, which is then phenol extracted. Add lithium chloride and isopropanol to precipitate the DNA. 4) Centrifuge and wash the DNA pellet with ethanol before resuspending it in a small volume of buffer.

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    This document provides a protocol for isolating genomic DNA from bacteria using triton purification. The key steps are: 1) Grow bacterial colonies in large-scale cultures and harvest the cells into a tube. 2) Resuspend the pellet in STET buffer and add a lysozyme/RNase mixture. Boil the mixture for 1 minute and 15 seconds. 3) Centrifuge the mixture and collect the supernatant, which is then phenol extracted. Add lithium chloride and isopropanol to precipitate the DNA. 4) Centrifuge and wash the DNA pellet with ethanol before resuspending it in a small volume of buffer.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as DOCX, PDF, TXT or read online from Scribd
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    204 views1 page

    Genomic DNA Isolation From Bacteria

    Uploaded by

    arvind_bmsresearchfellow
    This document provides a protocol for isolating genomic DNA from bacteria using triton purification. The key steps are: 1) Grow bacterial colonies in large-scale cultures and harvest the cells into a tube. 2) Resuspend the pellet in STET buffer and add a lysozyme/RNase mixture. Boil the mixture for 1 minute and 15 seconds. 3) Centrifuge the mixture and collect the supernatant, which is then phenol extracted. Add lithium chloride and isopropanol to precipitate the DNA. 4) Centrifuge and wash the DNA pellet with ethanol before resuspending it in a small volume of buffer.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as DOCX, PDF, TXT or read online from Scribd
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    Genomic DNA Isolation from Bacteria

    Arvind Singh*, Sushila Kala

    Biomedical Sciences Department, Bundelkhand University, Jhansi, India

    arvind_bmsresearchfellow@gmail.com

    Protocol:

    Preparation method for the isolation of geneomic DNA from bacteria using triton purification

     Grow several colonies - large scale-15ml cultures.


     Harvest the cells into a single eppendorf tube or a 15 ml disposable tube depending on the volume.
     Resuspend pellet with 300ul STET buffer (900ul).
     After resuspending add 30ul RNase/lysozyme mixture (100ul).
     Boil pellet for 1 minute 15 seconds (one minute 45 seconds).
     Spin in microfuge for 15 minutes.
     Take supernatant and phenol extract with 150ul (500ul) STET- saturated phenol.
     Spin and take supernatant. Add 1/10 volume 4M lithium chloride (autoclaved). On ice for 5-10 minutes.
     Spin and take supernatant. Add equal volume isopropanol. RT for 5 minutes.
     Spin.
     Wash with 80% ethanol (95% will cause the residual Triton to precipitate)
     Resuspend pellet in 50-200ul.
     Lysozyme/ RNase mixture 10mg/ml lysozyme 1mg/ml RNase (use cheap grade (BMB) rather than
    RNase A , which is too expensive) 50mM Tris-HCl pH8.0 Store at -20oC in small aliquots. Do not
    refreeze after thawing.
     STET 8% sucrose 5% Triton X-100 50mM Tris-HCl (pH8.0) 50mM EDTA pH 8.0 Filter sterilize. Store at
    4oC

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