Miniprep Protocol

1. Inoculate 3ml LB medium (containing antibiotic) with a bacterial clone, culture with vigorous shaking at 37 degree for 14-16 hrs. 2. Aliquot 2 x 750 ul culture into microcentrifuge tube, harvest bacteria by spinning at 12000rpm (~11000g) for 1 min. Aspirate supernatant. Add an additional 750 ul culture media, respin and aspirate supernatant. 3. Resuspend bacterial pellet by complete vortexing in 100ml of SOLUTION A. The bacteria should be completely resuspended - no clumps should be visible. Solution A is stored in the refrigerator. 4. Add 100ul freshly prepared LYSIS BUFFER (50ul 400mM NaOH, 50ul 2% SDS) and mix gently by inverting 5-6 times at room temperature. The mixture should appear translucent and mucous-like. 5. Add 120ul SOLUTION K (5M potassium acetate, pH5.5) and mix gently by inverting 5-6 times, incubate at room temperature for 3 min. This solution is kept in the refrigerator. The mixture should contain flocculent white precipitate at this point. 6. Remove bacterial debris by centrifugation at 12000rpm for 2 min, transfer supernatant to a fresh microcentrifuge tube. The precipitate is "sticky". To transfer use a 1 ml pipet tip, depress pipet plunger, move tip to bottom of tube and aspirate supernatant. When transfering to new tube, do not touch outside of pipet tip to new tube - avoid transfer of precipitate to the new tube. 7. Add 200ul isopropanol (2-Propanol) to precipitate plasmid DNA from supernatant. Mix thoroughly by inverting 10 times. Incubate at room temperature for 1 min. 8. Precipitate DNA by centrifugation at 14000 rpm for 1 min. Pour off isopropanol into beaker. 9. Add 500ul 70% ethanol to tube, mix by inverting (brief vortexing okay too). Spin the DNA down at 14000rpm (full speed) for 1min. 10. Pour off ethanol. Respin for 1 min. Use 100 ul pipet to carefully remove remaining ethanol. Air dry DNA for 10 min. 11.Dissolve DNA in 100 ul of TE buffer.

BUFFERS NEEDED
BUFFER A: 25mM Tris-HCl, 10mM EDTA, pH8.0 LYSIS BUFFER: Mix of 400mM NaOH and 2% SDS (1:1) SOLUTION K: 5M potassium acetate, pH5.5 TE BUFFER: 10 mM Tris-Cl, 1 mM EDTA, pH7.5 ISOPROPANOL and 70 % ETHANOL