• 圣淘沙名胜世界(新加坡)

    Hybridization and Immobilization

    Chia-Hsien Shih

    Published: 2021-10-18 DOI: 10.17504/protocols.io.by5gpy3w
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    Abstract

    This protocol is to assemble GotCha with circular probe, immobilization probe and magnetic beads.

    Steps

    Preparation

    1.

    Dilute immobilization probe into 10µM

    Protocol of Hybridization

    2.

    Add 4.5µL of 1µM circular probe

    3.

    Add 9µLof 10X 7.4 PBS buffer

    4.

    Add 0.9µL of 10µM immobilization probe

    5.

    Add 75.6µL RNase-free water

    6.

    Vortex slightly and spin down

    7.

    Heated to 90°C for 0h 0m 30s then cool down to 90Room temperature and keep for 0h 30m 0s

    Protocol of Immobilization

    8.

    Take 22.5µL of 10mg/ml magnetic beads into a eppendorf

    9.

    Vortex then spin down in Centrifuge 15000rpm

    10.

    Carefully take the eppendorf onto the DynaMag and wait for 0h 1m 0s to collect the beads

    11.

    Wash beads with 1X PBS for three times. Make sure that eppendorf should put on DynaMag when removing supernatant.

    12.

    Add 90µL of hybridization solution into the eppendorf with dry beads

    13.

    Incubate at 90Room temperature for 0h 30m 0s, then spindown in Centrifuge15000rpm

    14.

    Carefully take the eppendorf onto the DynaMag and wait for 0h 1m 0s to collect the beads

    15.

    Wash beads with 1X PBS for three times. Make sure that eppendorf should put on DynaMag when removing supernatant.

    16.

    Preserve the functional beads in 90µL 1X PBS

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