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    Primer For Sequencing Prefer Tm: 50-60C, GC content: 40-60%

    E Coli Colonies

    • Colonies need to grow for at least 16hrs at 37C to reach good visible size
    • Pick a single colony with sterile tip and resuspend in 30ul sterile Tris buffer (10mM, ph8.0)
    • Prepare 2 separate tubes, 1 contains 15ul colony suspension, 1 contains 5ul primer at 5pmol/ul

    Plasmid Walking

    • 10ug plasmid DNA
    • Vector information if whole plasmid sequencing to be done
    • 10ul vector primers for first round of sequencing at 5uM
    • Reference or expected sequence if any