Preparation of pPSU 100 bp and 1 kb ladders. This protocol yields 2.5 mL of each ladder — scale accordingly.

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Image path corrected during migration. The ladder key image was linked as ../img/… in the source, one directory level too high, so it did not display on the lab docs site.

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  1. Mix the following in 1.5 mL tubes.

    EcoRV digest (1 kb ladder)

    Reagent Amount
    10X rCutSmart Buffer 20 μL
    pPSU1 50 μg
    pPSU2 40 μg
    20 units/μL EcoRV-HF 4 μL
    Water To 200 μL

    PstI digest (100 bp ladder)

    Reagent Amount
    10X rCutSmart Buffer 20 μL
    pPSU1 50 μg
    pPSU2 40 μg
    20 units/μL PstI-HF 4 μL
    Water To 200 μL

  2. Digest overnight at 37°C.

  3. Check 0.5 μL of digest on a 1% agarose gel. Confirm the ladder is the correct configuration using the key below, for both the 100 bp and 1 kb ladders.

    pPSU ladder key

    pPSU ladder key

  4. Mix the following in a 15 mL Falcon tube.

    1 kb ladder stock

    Reagent Amount
    10 mM Tris-Cl pH 8.0, 0.1 mM EDTA 1.98 mL
    EcoRV digest 0.1 mL
    6X gel loading buffer 0.42 mL

    100 bp ladder stock

    Reagent Amount
    10 mM Tris-Cl pH 8.0, 0.1 mM EDTA 1.98 mL
    PstI digest 0.1 mL
    6X gel loading buffer 0.42 mL

  5. Make 500 μL aliquots in 1.5 mL tubes. Keep 1 aliquot at 4°C for regular use and store the remaining aliquots in the -20°C freezer.