Preparation of Penn State DNA ladders (pPSU) - MetabolicEngineeringGroupCBMA/MetabolicEngineeringGroupCBMA.github.io GitHub Wiki

This procedure is based on the CAVAN low cost plasmid miniprep protocol without kit (super snik) and uses the same solutions NEVER MIX pPSU1 AND pPSU2 SUSPENSIONS/SOLUTIONS UNTIL AFTER ENZYMATIC DIGESTION!

Ladder preparation:

• Inoculate each XL1-BLue colony (pPSU1 and pPSU2) in 50 mL of LB+amp O/N 37°C shaking (2 250 mL elrnemeyer flaks)
• Transfer each suspension to a 50 mL falcon and spin down at 5000 RPM for 10 min
• Resuspend each pellet with 3 mL of Resuspension solution (vortex)
• Aliquot each suspension in 600 µL volumes in 2 mL tubes (10-12 tubes for each plasmid)
• Add 600 µL Lysis solution and invert 3X (do NOT vortex)
• Incubate for ~3 min 30 s (no longer than 5 minutes!)
• Add 600 µL Neutralization solution and invert 3X (do NOT vortex)
• Incubate up to 5 minutes
• Spin down for 30 min at 14000 RPM at 4°C
• Aliquot supernatant in 1 mL volumes in 2 mL tubes (~20 tubes each plasmid)
• Add 1 mL of Isopropanol (precipitation)
• Vortex and incubate 2 min at RT
• Spin down for 1 h (14000 RPM)
• Discard supernatant
• Wash pellet with 500 µl of 70% EtOH
• Spin down at 4°C for 5 min at 14000RPM
• Discard supernatant
• Repeat washing step
• Dry pellet with speedvac for 30 min (or overnight to the air)
• Add 50 µL of ultra pure H2O
• Mix all 50 µL aliquots of the same plasmid into the same 2mL tubes with 1 mL for each tube (DON'T mix pPSU1 and pPSU2)
• Add 790 µL of ultra pure H2O to each tube
• Add 200 µL of the apropriate digestion buffer to each tube
• Add 10 µL of either EcoRV or NcoI(or BglII) to each pPSU1 tube and to each pPSU2 tube
• Vortex and incubate 36 h at 37°C
• Inactivate restriction enzymes by incubating 30 min at 80ºC
• In a 2 mL tube, mix 250 µL of EcoRV digested pPSU1, 250 µL of EcoRV digested pPSU2,250 µL of NcoI(or BglII) digested pPSU1 and 250 µL of NcoI(or BglII) digested pPSU2. Add 1 mL of loading buffer (2X or of greater concentration)

Plasmids pPSU1 (10kb) and pPSU2 (7.75kb) are two plasmids that can be used to create DNA molecular weight markers (DNA ladders) for size comparisons on agarose or polyacrylamide gels.

These plasmids are described here Henrici RC, Pecen TJ, Johnston JL et al. The pPSU Plasmids for Generating DNA Molecular Weight Markers. Sci Rep 2017;7:2438.

A mixture of both plasmids cut with PstI produces a 100 bp ladder while digestion with EcoRV yields a 1 kb ladder.

Preparation-of-Penn-State-DNA-ladders