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Preparation of pPSU 100 bp and 1 kb ladders

This protocol yields 2.5 mL of 100 bp and 1 kb ladder, each. Scale accordingly.

  1. Mix the following in 1.5 mL tubes:

    Reagent Amount
    10x CutSmart Buffer 20 μL
    pPSU1 50 μg
    pPSU2 40 μg
    20 units/μL EcoRV-HF 4 μL
    Water To 200 μL
    Reagent Amount
    10x CutSmart 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 100 bp and 1kb ladders.

  1. 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
  2. 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.