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SPEEDY CHEAP PLASMID PREPS 1. Spin down cells in 1.5 mL eppendorf tube at 9,000 rpm for 30s. Pour off supernatant into waste beaker, repeat until all cells are spun. Quick spin the last bit of media and pipet off. 2. Add 100 μL Resuspension Buffer and resuspend cells completely by pipetting or vortexing. 3. Add 200 μL Lysis Buffer. Invert until solution is clear, no longer than 5 minutes. 4. Add 150 μL 7.5 M ammonium acetate. Vortex and place on ice 5 minutes. 5. Spin cellular debris down for 10 minutes at max speed. 6. Precipitate plasmid DNA Remove supernatant to 800 μL 95% ethanol. Invert tubes to mix, leave at room temp 1 minute. Spin for 3 minutes at max speed. Pour off ethanol (you should see a pellet). Add 400 μL 70% ethanol (to wash the pellet). Quick spin, pour off 70% ethanol. Quick spin, pipet off remaining liquid (avoiding the pellet). Resuspension Buffer (aka P1 Buffer) Lysis Buffer (aka P2 Buffer) 50 mM Tris, pH 8 0.2 N NaOH 10 mM EDTA, pH 8 1% SDS 100 μg/mL RNase A
"SPEEDY CHEAP PLASMID PREPS"