1. Inoculate 1-5 ml LB medium (selective antibiotic added) with a single colony, incubate with vigorous shaking at 37 °C overnight. 2. Harvest bacteria by centrifuging at > 6000 rpm for 5 min. Remove supernatant and keep pellet. 3. Resuspend bacterial pellet with 250 μl Buffer P1 and transfer to a fresh microcentrifuge tube. 4. Add 250 μl Buffer P2 and mix gently by inverting 5-6 times at room temperature. 5. Add 350 μl Buffer P3 and mix gently by inverting 5-6 times. Spin down at 12000 rpm for 10 min to remove bacterial/protein debris. Transfer supernatant to a spin column. 6. Centrifugation at 6000 rpm for 1 min. Discard flow-through.7. (Optional), wash spin column with 0.5 ml Buffer PB; centrifuge at 6000 rpm for 1 min, and discard flow-through.8. Wash spin column with 0.75 ml Buffer PE; centrifuge at 6000 rpm for 1 min, and discard flow-through.9. Centrifuge at 12000 rpm for an additional 1 min to remove residual wash buffer.10. To elute DNA, place the column in a clean 1.5 ml collection tube. Add 50 μl Buffer EB to the center of the membrane, let stand for 1 min, and centrifuge at 12000 rpm for 1 min to collect eluted DNA.