Team:Lyon-INSA-ENS/Realisation/Protocols/Miniprep

Plasmid DNA purification using QIAprep® Spin Miniprep Kit

This protocol is designed for purification of up to 20 µg of high-copy plasmid DNA from 1-5mL overnight cultures in LB mediul.

Procedure

1. Pellet 1-5mL bacterial overnight culture by centrifugation at >8000 rpm ( we used 13000 ) for 3mn at room temperature

2. Resuspend pelleted bacterial cells in 250µL buffer P1 and transfer to a microcentrifuge tube

3. Add 250µL buffer P2 and mix thoroughly by inverting the tybe 4-6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5mn.

4. Add 350µL buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.

5. Centrifuge for 10mn at 13000 rpm in a table-top microcentrifuge

6. Apply the supernatant from step 5 to the QIAprep spin column by decanting or pipetting. Centrifuge for 30-60s and discard the flow-through.

7.Recommended if the strain is endA+ Wash the QIAprep spin column by adding 0.5mL buffer PB. Centrifuge for 30-60s and discard the flow-through.

8. Wash the QIAprep spin column by adding 0.75 mL buffer PE. Centrifuge for 30-60s and discard the flow-through.

9. Centrifuge for 1mn to remove residual wash buffer

10. Place the QIAprep column in a clean 1.5mL microcentrifuge tube. To elute DNA, add 50 µL buffer EB ( 10mM Tris.Cl, pH 8.5 ), let stand for 1mn and centrifuge for 1mn.

This protocol is extracted from "QIAprep Miniprep Handbook" by QIAGEN .