1. Isolate a single colony from a freshly streaked selective plate, and inoculate a culture of 5mL LB medium containing the appropriate selective antibiotic. Incubate for12~16 hours at 37℃ with vigorous shaking(180rpm). Centrifuge at 13,000 x g for 1 minute at room temperature.

2. Add 250μL Solution I, vortex or pipet up down to mix thoroughly.

3. Add 250μL Solution II, invert and gently rotate the tube several times to obtain a clear lysate. A 2-3 minute incubation may be necessary.

4. Add 350μL Solution III, immediately invert several times until a flocculent white precipitate forms I.

5. Centrifuge at maximum speed(13,000 x g) for 10 minutes. A compact white pellet will form. Promptly proceed to the next step. Insert a HiBind DNA Mini Column into a 2mL Collection Tub.

6. Transfer the cleared supernatant from Step 5 by carefully aspirating it into the HiBind DNA Mini Column. Be careful not to disturb the pellet and that no cellular debris is transferred to the HiBind DNA Mini Column. Centrifuge at maximum speed for 1 minute. Discard the filtrate and reuse the collection tube.

7. Add 500μL HBC Buffer.Centrifuge at maximum speed for 1 minute. Discard the filtrate and reuse collection tube.

8. Add 700μL DNA Wash Buffer. Centrifuge at maximum speed for 1 minute. Discard the filtrate and reuse the collection tube.

9. Centrifuge the empty HiBind DNA Mini Column for 2 minutes at maximum speed to dry the column matrix.

10. Transfer the HiBind DNA Mini Column to a clean 1.5mL microcentrifuge tube. Add 30μL Elution Buffer or sterile deionized water directly to the center of the column membrane.

11. Store DNA at -20°C.