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.
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Mix the following in 1.5 mL tubes:
Reagent Amount 10x rCutSmart Buffer 20 μL pPSU1 50 μg pPSU2 40 μg 20 units/μL EcoRV-HF 4 μL Water To 200 μL Reagent Amount 10x rCutSmart Buffer 20 μL pPSU1 50 μg pPSU2 40 μg 20 units/μL PstI-HF 4 μL Water To 200 μL -
Digest overnight at 37°C.
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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.
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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 -
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.