How do I use silica powder to prepare plasmid DNA for sequencing?
This method from Bill Melchior [wmelchior@ntbtox.nctr.fda.gov] is based on modifications of the Birnboim and Doly alkaline lysis method. For more information, see Birnboim1979, Birnboim1983. Morelle1989, and Carter1993. MATERIALS: • 50 mM glucose/10 mM EDTA/25 mM Tris (pH 8) (“GET”)…store in refrigerator. • RNase A 10, mg/ml, heat treated…store frozen. • 0.2 N NaOH/1% SDS…store at room temperature. • 7.5 M ammonium acetate…store refrigerated.. • Silica slurry: 50% solution of powdered silica in H20…store refrigerated or frozen. • NaI/Na2SO3 (Na sulfite)…store at room temperature. • NEET wash solution…store in freezer. (Note: The preparation of the last three items is described in Question 11). PROCEDURE: • Grow 3 ml overnight cultures of plasmid-containing bacteria. • After chilling overnight cultures on ice for 5 minutes, collect the bacteria in 1.5 ml microcentrifuge tubes by centrifugation. After decanting, centrifuge the tubes 10 seconds and aspirate the remaining liq
