Plasmid Miniprep with Alkaline Lysis

Sambrook and Maniatis

1. Spin down O/N culture in 1.5 mL microfuge tube.
2. Reuspend pellet in 100 uL Solution I.
3. Add 200 uL of fresh Solution II.
4. For 1 mL Solution II: 2 uL 10 N NaOH + 50 uL 20% SDS + 948 uL dH2O
5. Invert tube 4-6 times. Store on ice.
6. Add 150 uL Solution III. Invert tube to mix.
7. Centrifuge 10-12 min. at 12,000 RPM in the microcentrifuge Transfer supernatant (400 mL) to a new tube.
8. Add 400 uL phenol/chloroform. Mix well by vortexing. Spin 3 min. Transfer aqueous (TOP!!) layer to new tube.
9. Precipitate DNA by adding 800 uL 95% EtOH. Chill at -20 C for 10 minutes, spin at 4C, 12,00 RPM.
10. Wash pellet with 500 uL of 70% ethanol. Spin at 12,000, 4C for 5 minutes.
11. Pour off supernatant and allow pellet to dry completely.
12. Resuspend DNA in 40 uL TE/RNase or sterile dH2O.