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    Cost-efficient Yeast genome Flongle library 

    Yutaro Hori

    Published: 2022-12-16 DOI: 10.17504/protocols.io.e6nvwjb2wlmk/v1
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    Abstract

    A cost-efficient protocol for constructing a Flongle library.

    Steps

    1.

    Assemble the following components to end repair and add A to the genomic sample:

    AB
    Ultra II buffer1.75
    End repair buffer1.75
    Ultra II enzyme1
    End repair enzyme1
    DNAx (500 ng)
    MQ44.5 - x

    Incubate at 20°C for 0h 5m 0s, then 65°C for 0h 5m 0s.

    2.

    Add 50µL of Ampure beads (or equivalent beads) and mix well.

    Wash with 75 % EtOH twice and elute with 11µL of 10millimolar (mM) Tris-HCl (pH 8.0).

    Keep the tube on the magnetic rack and do not disturb the beads while washing with 75% EtOH.

    Use 1µL of the solution to check the concentration with Qubit. The concentration needs to be at least 30ng/uL.

    3.

    Assemble the following components for ligation:

    AB
    DNA10 uL
    TLB4 uL
    Quick ligase1.5 uL
    AMX0.8 uL

    Incubate at Room temperature for 0h 30m 0s.

    Add 2.3µL of 5Molarity (M) NaCl and incubate at Room temperature for 0h 30m 0s and then cfg at max speed for 0h 20m 0s.

    Remove sup and add PEG wash buffer, cfg at max speed for 0h 1m 0s .

    Remove sup and add 11µL of 10mM Tris-HCl (pH 8.0) and incubate at 4°C for.

    Check the concentration with Qubit.

    4.

    Assemble the following priming solution:

    AB
    FLB2.5 uL
    FB97.5 uL

    and the library mix solution:

    AB
    DNAx uL (100 ng)
    SB II10 uL
    LB II4 uL
    MQ6 - x uL

    . Load them and start sequencing.

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