• 黄金城集团

    HTTM : gDNA extraction

    Antoine Champie, Amélie De Grandmaison, Sebastien Rodrigue

    Published: 2024-05-22 DOI: 10.17504/protocols.io.q26g7yzz3gwz/v3
    AI 解读
    浏览 1346

    Abstract

    Part of the HTTM protocol dedicated to the extraction of gDNA from transposon mutated cell pellets.

    Attachments

    HDTM _ Protocol-2.pdf

    Steps

    DNA extraction

    1.

    Prepare the lysis solution by adding 165µL of proteinase K to 66mL of homemade lysis buffer and mix well.

    2.

    Add 600µLof lysis solution to each well of the deep-well plate and resuspend the pellet.

    3.

    Cover with an adhesive aluminum foil and incubate at 55°C for 1h 0m 0s.

    4.

    While still warm, add 260µLof ethanol 100%, without overmixing.

    Note
    Overmixing will result in DNA agglomeration and difficulty with the extraction.

    5.

    Transfer immediately to a deep-well plate fitted with an array of silica columns.

    6.

    Centrifuge twice at 3270x g .

    7.

    Discard flowthrough and add 500µL of wash solution.

    8.

    Centrifuge at 3000x g

    8.1.

    Repeat steps 7 and 8.

    9.

    Discard flowthrough.

    10.

    Centrifuge at 3000x g to eliminate traces of wash solution.

    11.

    Discard flowthrough.

    12.

    Add a collector plate between the silica column array and the deep-well plate.

    13.

    Add 50µLof low TE to the silica matrix in each well.

    14.

    Cover with an adhesive aluminum foil and incubate at55°C for 0h 15m 0s.

    15.

    Centrifuge at 3270x g .

    Silica array regeneration (Optional)

    16.

    Put the contaminated silica array on an empty deep-well plate.

    Add 150µL of 1N NaOH + 0.15%(v/v) Triton X-100 to each well.

    17.

    Incubate at Room temperature for 0h 5m 0s

    18.

    Centrifuge 3000x g

    19.

    Add 200µL of 1,5N HCl+ 0,15% (v/v) Triton X-100 to each well.

    20.

    Incubate at Room temperature for 0h 30m 0s

    21.

    Centrifuge 3000x g

    22.

    Add 150µL of 1N NaOH + 0,15%(v/v) Triton X-100 to each well.

    23.

    Incubate at Room temperature for 0h 5m 0s

    24.

    Centrifuge 3000x g

    24.1.

    Collect the flowthrough in a beaker. Neutralize pH if needed and dispose of the flow through.

    25.

    Add 600µLof ddH2O to each well.

    26.

    Centrifuge 3000x g

    27.

    Silica columns array are ready to be reused.

    推荐阅读

    Nature Protocols
    Protocols IO
    Current Protocols
    扫码咨询