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2.3.1.11: Linearizing Backbone Supplies

  • Page ID
    114852
    • Nathan Reyna, Ruth Plymale, & Kristen Johnson
    • Ouachita Babtist University & University of New Hampshire

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    Linearizing Backbones:

    Materials

    Amount Needed: (Per Backbone Reaction)

    Q5 High-Fidelity 2X Master Mix (Cat #: M0492S)

    Up to 25 uL

    10 uM Forward Primer (SB-prep-3P-1)

    Up to 2.5 uL

    10 uM Reverse Primer (SB-prep-2Ea)

    Up to 2.5 uL

    Template DNA (psB1C3, A3, K3, T3)

    10 ng

    Distilled Water

    Up to 60 uL

    Thermocycler

    1

    UV Spectrophotometer

    1

    Micropipette and Tips

    1-10 uL, 0.2-1 uL, 20-200 uL

    Heat Block (80C)

    1

    Gel Rig

    1

    PCR Clean-up Kit (Cat #: D4033)

    1

    10X NEB Buffer 2.1

    4 uL

    Digestion Enzymes (EcoRI, PstI, DpnI)

    1 uL of each

    Competent Bacteria Cells

    (JM109= Cat#: T3003)

    25 uL

    LB Agar Plate with Antibiotic (Chlor, Amp, Tet, Kan)

    1 plate (depending on backbone)

    Sterile Glass Beads

    Approximately 4-8 beads

    SOC Media (Cat#: 15544034)

    200 uL

    Shaking Incubator (37C)

    1

    T4 DNA ligase (Cat#: M0202S)

    0.5 uL

    T4 DNA Ligase Buffer(Cat#: B0202S)

    1 uL


    This page titled 2.3.1.11: Linearizing Backbone Supplies is shared under a not declared license and was authored, remixed, and/or curated by Nathan Reyna, Ruth Plymale, & Kristen Johnson.