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Plasmid Isolation Protocol

• Following is a plasmid isolation protocol provided by QIAGEN:

1. Resuspend pelleted bacterial cells in 250µl Buffer P1 and transfer to a microcentrifuge tube
2. Add 250µl Buffer P2 and mix thoroughly by inverting the tube 4-6 times
3. Add 350µl Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times
4. Centrifuge for 10 min. at 13,000 rpm in a table-top microcentrifuge
5. Apply the supernatant (from step 4) to the QIAprep spin column by decanting
6. Centrifuge for 30-60 seconds and discard the flow-through
7. Wash QIAspin column by adding 0.75ml Buffer PE and centrifuging for 30-60 seconds
8. Discard the flow-through, and centrifuge for an additional 1-3 minutes to remove residual wash buffer
9. To elute DNA, place the QIAprep column in a clean 1.5ml microcentrifuge tube. Add 50µl water to center of QIAprep spin column, let stand for 1 minute and centrifuge for 1-3 minutes.

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