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    SHERLOCK protocol, updated for sharing
    • Notes
    • Metadata
    • Relevant Items
    • in vitro transcription of crRNAs
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    SHERLOCK protocol, updated for sharing

    Tuesday, 11/20/2018Buffer compositionsCleavage buffer (Tris, final pH 7.5)Table2Storage bufferTable1RPA reactionReaction setupLwaCas13a detectionMaster Mixes
    Tuesday, 11/20/2018
    Buffer compositions
    Cleavage buffer (Tris, final pH 7.5)
    Storage buffer
    RPA reaction
    Components needed
    ●
    TwistDx RPA kit
    ●
    RPA primers
    ●
    Sample
    Each pellet makes enough reaction for 4 samples
    1.
    Make master mix (all components except MgAc). In this case pre-diluted 10uM primers were used.
    2.
    Resuspend RPA pellets with 1 part master mix (30uL). Resuspend pellet (keep tip) and add back to master.
    3.
    Add MgAc.
    4.
    Aliquot 10uL of master mix to A1-6
    5.
    Add 1uL per sample (see above for layout in wells) into 10uL.
    6.
    Cover in heat-resistant clear top.
    7.
    Run in thermocycler at 37 C for 30 min-hour.
    LwaCas13a detection
    Components needed
    ●
    crRNA (300ng/uL)
    ●
    NEB rNTP (NEB N0466L)
    ●
    Lucigen T7 polymerase (Lucigen 30223-2)
    ●
    NEB Murine RNA inhibitor (NEB M0314L)
    ●
    MgCl2 (Thermo AM9530G)
    ●
    Storage buffer
    ●
    Cleavage buffer (
    ●
    Background RNA (harvested from HEK293FT mammalian)
    ●
    384 well plate (Corning 3542)
    IDT brown tube of RNaseAlertv2 is 2nmol. Resusepnd in 1mL of water for 2uM final.
    Protein is a 1:31.7 dilution from 2mg/mL stock. Resuspended 4uL of protein in 122.5uL of Storage Buffer.
    Read on a plate reader in a 384 well plate (20uL/well) every 5 minutes at FAM excitation/emission (495/520).

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