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    Viral purification from bacterial culture

    sarah.schulz

    Published: 2023-03-16 DOI: 10.17504/protocols.io.e6nvwjq7wlmk/v1
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    Abstract

    Protocol for the purification of viral particles from bacterial liquid culture

    Steps

    1.

    Centrifuge 40 ml of bacterial culture infected with phage at 6000 g for 30 min, remove the supernatant and filter it through a 0.45 μm syringe filter

    2.

    Centrifuge the filtrate at 35 000 g for 4 h, remove the supernatant and re-suspend the pellet in 600 μl SM buffer

    3.

    Add 2 μl of DNAse I and 20 μl of 10x DNAse buffer and incubate at 37°C for 1.5 h

    4.

    Incubate sample at 65°C for 30 min to inactivate DNAse I

    5.

    Add 10 μl of 20 % SDS and 40 μl of Proteinase K (20 mg/ ml) and incubate at 37°C for 1 h

    6.

    After the incubation, mix the sample with an equal amount of phenol:chloroform:isoamyl pH 8.0 (25:24:1) alcohol in a phase lock gel light tubes and centrifuge at 12 000 g for 5 min

    7.

    Add 600 μl more of the phenol:chloroform:isoamyl alcohol to the tube and centrifuge at 12 000 g for 5 min

    8.

    Transfer the aqueous phase to a new tube and add 1200 μl cold 100% ethanol

    9.

    Incubate sample overnight at -80°C, and then centrifuge at 16 000 g for at 4°C for 1 h

    10.

    Remove the supernatant and re-suspend the pellet in 100 μl TE buffer

    11.

    Store at 4°C

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