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    Nib Dna Extraction Protocol

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    preethi.elizabeth2022
    The document outlines a protocol for extracting DNA from bacterial cultures using an extraction buffer to isolate the DNA, which is then dissolved in TE buffer. Key steps include pelleting the bacterial culture, adding extraction buffer containing sucrose, EDTA, Tris-HCl, NH4Cl, Triton X-100, lysozyme, RNase and CaCl2, incubating at 65 degrees Celsius, centrifuging to separate supernatant containing DNA, precipitating DNA with isopropanol and washing with ethanol before dissolving extracted DNA in TE buffer. The extracted DNA can then be run on a gel to assess quality and quantity.

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    Nib Dna Extraction Protocol

    Uploaded by

    preethi.elizabeth2022
    14 views1 page
    The document outlines a protocol for extracting DNA from bacterial cultures using an extraction buffer to isolate the DNA, which is then dissolved in TE buffer. Key steps include pelleting the bacterial culture, adding extraction buffer containing sucrose, EDTA, Tris-HCl, NH4Cl, Triton X-100, lysozyme, RNase and CaCl2, incubating at 65 degrees Celsius, centrifuging to separate supernatant containing DNA, precipitating DNA with isopropanol and washing with ethanol before dissolving extracted DNA in TE buffer. The extracted DNA can then be run on a gel to assess quality and quantity.

    Original Title

    16. NIB DNA EXTRACTION PROTOCOL

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    The document outlines a protocol for extracting DNA from bacterial cultures using an extraction buffer to isolate the DNA, which is then dissolved in TE buffer. Key steps include pelleting the bacterial culture, adding extraction buffer containing sucrose, EDTA, Tris-HCl, NH4Cl, Triton X-100, lysozyme, RNase and CaCl2, incubating at 65 degrees Celsius, centrifuging to separate supernatant containing DNA, precipitating DNA with isopropanol and washing with ethanol before dissolving extracted DNA in TE buffer. The extracted DNA can then be run on a gel to assess quality and quantity.

    Copyright:

    © All Rights Reserved
    Download as DOCX, PDF, TXT or read online from Scribd
    Download as docx, pdf, or txt
    14 views1 page

    Nib Dna Extraction Protocol

    Uploaded by

    preethi.elizabeth2022
    The document outlines a protocol for extracting DNA from bacterial cultures using an extraction buffer to isolate the DNA, which is then dissolved in TE buffer. Key steps include pelleting the bacterial culture, adding extraction buffer containing sucrose, EDTA, Tris-HCl, NH4Cl, Triton X-100, lysozyme, RNase and CaCl2, incubating at 65 degrees Celsius, centrifuging to separate supernatant containing DNA, precipitating DNA with isopropanol and washing with ethanol before dissolving extracted DNA in TE buffer. The extracted DNA can then be run on a gel to assess quality and quantity.

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    © All Rights Reserved
    Download as DOCX, PDF, TXT or read online from Scribd
    Download as docx, pdf, or txt
    of 1

    NIB DNA EXTRACTION PROTOCOL

    Purpose – for extracting the DNA from a bacterial culture for further use in PCR and other
    reactions and purposes. Here extraction buffer is used to isolate dna and then dissolved in TE
    buffer.
    Chemicals used – bacterial mother culture fresh, TE buffer (pH 8), sucrose (5%), 50 mm edta, 50
    mm tris hcl (ph 8), 0.5% triton x, 0.75 m nh4cl, 100 ug/ml lysozyme, 25 ug/ml rnase a, 50 mm
    cacl2.
    Materials – cold centrifuge, cold isopropanol and IPA, TE buffer, autoclaved water,
    electrophoresis, agarose gel, geldoc.
    Procedure –
    1. Pellet 2 ml of the fresh bacterial mother culture.
    2. Centrifuge at 10k rpm for 8 minutes in RT. Repeat to get more pellet.
    3. Prepare extraction buffer with 2.5 g sucrose, 5 ml edta (50mm), 2.5 ml tris hcl (50 mm), 2
    g nh4cl, 250 ul triton x, 5 mg lysozyme, 100 ul rnase, 367.5 mg cacl2 (50 mm).
    4. Keep in fridge after mixing well.
    5. To the bacterial pellet add 150 ul of the extraction buffer.
    6. Mix and vortex well.
    7. Incubate at 65 degrees for 5 mins in dry bath.
    8. Spin at 12k rpm for 10 mins.
    9. Discard the pellet.
    10. Add 120 ul of IPA to supernatant and mix well and incubate at RT for 15 mins.
    11. Spin for 12k rpm for 10 mins.
    12. Discard the supernatant, and add 1 ml 70% etoh and wash.
    13. Air dry and dissolve in 20 – 50 ul TE buffer.
    Results – you will get the gel with dna and calculate ng/ul with a ladder reference.
    Reagents –
    1. Extraction buffer
    2. TE buffer
    3. IPA

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