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    Viral PEG purification

    sarah.schulz

    Published: 2023-03-16 DOI: 10.17504/protocols.io.n92ldp6dxl5b/v1
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    Abstract

    Protocol for viral particle purification via PEG percipitation

    Steps

    1.

    Add 30 ml of ice cold PBS to 3 g of faecal sample and mix by vortexing at high speed for 5 min, then keep on ice

    2.

    Centrifuge sample at 5000 rpm for 10 min at 4°C and transfer the supernatant to a clean 50 ml centrifuge tube

    3.

    Filter the supernatant through a 0.45 μm syringe filter

    4.

    Add 10 % 10% w/v PEG-8000 in saline (0.5 M NaCl) to filtrate, mix by inverting and incubate at 4°C overnight

    5.

    Centrifuge sample at 5000 rpm for 20 min at 4°C and discard the supernatant.

    6.

    Re-suspend pellet in 200 μl PBS and transfer it to a 1.5ml Eppendorf tube

    7.

    Add an equal volume of chloroform to the sample and mix gently before centrifuging it at 2500 rpm for 5 min at room temperature

    8.

    Transfer the aqueous phase into a new and sterile 1.5 ml tube

    9.

    Add 40 μl of 10 mM CaCl2 and 50 mM MgCl2 to the sample, as well as 10 U of DNase and 10 U of RNase A

    10.

    Incubate samples at 37°C for 2 h and then place them in a heatblock at 70°C for 10 min

    11.

    Store at 4°C

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