We use Wizard minipreps ONLY when we are planning to make RNA from a plasmid or you are doing particularly tricky subcloning, but not for standard plasmid characterization!

Grow up overnight culture of bacteria in LB + ampicillin (50-100 µL from 100mg/ml stock at 37°C.

• Spin down 1.5 mL cells in microfuge tube, setting 5 on small microfuges (all steps at room temperature).
• Discard supernatant (if the culture is sparse, you can add another 1.5 ml and centrifuge again -- but remember to keep ~1ml of the culture in case you want to make a DMSO stock.
• Resuspend cells in 200 µL resuspension buffer (by running tube along a tube rack).
• Add 200 µL lysis buffer, invert to mix add 10µL of the alkaline protease. Let sit 10 minutes. mix only by inversion -- otherwise you will shear the chromosomal DNA
• Add 300 µL neutralization buffer, invert
• Spin 10 minutes at full speed
• Transfer supernatant to miniprep column, taking care to avoid disturbing the pellet and avoid "scum" on the surface (if possible).
• spin in microfuge for 1 min.
• Remove liquid from collection tube, add 0.5ml of wash buffer to column
• spin in microfuge for 1 min
• Remove liquid from collection tube, add 0.25ml of wash buffer to column
• spin in microfuge for 1 min
• Remove liquid from collection tube,
• spin in microfuge for 1 min
• discard collection tubes, place columns in 1.7ml microfuge tubes (with caps) and 100µL of water or Elution Buffer to each column (mark each tube).
• spin in microfuge for 1 min. (use one of the larger centrifuges, which can spin the tubes with their caps open).
• Discard column, cap tube.
• Check by gel electrophoresis - store in bag