1. evo视讯(中国)

    DNA extraction (BOMB)

    Yin-Tse Huang, Tsu-Chun Hung

    Published: 2022-07-04 DOI: 10.17504/protocols.io.261gen7ryg47/v1
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    Abstract

    DNA extraction (BOMB)

    Steps

    Sample Collection

    1.

    Add 200µL of 1mm beads to 1.5ml enppendorf tube

    2.

    Add200µL of 0.5mm beads to 1.5ml enppendorf tube

    3.

    Add 225µL of TE buffer to 2ml enppendorf tube

    Note
    TE buffer is in 4°C fridge

    4.

    Add 375µL of lysis buffer to 2ml enppendorf tube

    Note
    Lysis buffer is in 4°C fridge

    5.

    Add 267µL of 10M ammonium acetate to 2ml enppendorf tube

    Note
    10M ammonium acetate is in 4°C fridge

    6.

    Collect 10-20mg of sample to 2ml enppendorf tube

    Sample crush

    7.

    Put 2ml eppendorf tube in mixmill for sample crush, at this condition: 30 rpm/s, for 4mins 0h 4m 0s

    Centrifugation

    8.

    Put 2ml eppendorf tube in centrifuge for centrifugation, at this condition:17.0x g,25°C

    DNA purification

    9.

    Add 350µL of isopropanol to the 1st well of 96 well plate

    9.1.

    Add 125µL of magnetic beads (10 mg/ml) to the 1st well of 96 deep well plate

    Note
    Shake the bottle and pipetting before using magnetic beads

    9.2.

    Add 200µL of the sample (lysate) from the 2ml centrifuged tube to the 1st well of 96 deep well plate

    Note
    USUALLY ADD at LAST

    10.

    Add 400µL of isopropanol to the 2nd well of 96 deep well plate

    11.

    Add 300µL of 80% enthanol to the 3rd well of 96 deep well plate

    12.

    Add 300µL of 80% enthanol to the 4th well of 96 deep well plate

    13.

    Add 100µL of DD water to the 5th well of 96 deep well plate

    14.

    Put the prepared 96 deep well plate in the automated DNA extraction machine

    15.

    After the extraction is done, collect 100µL of the eluted sample as the DNA template for downstream experiments

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