Minipreps using the Qiagen kit

Plasmid extraction

Materials required

• Eppendorf tubes
• Pipettes
• Appropriate overnight cultures
• Buffer P1 (In the fridge)
• Buffer P2
• Buffer N3
• Buffer PB
• Buffer EB
• QIAprep spin column

Procedures

1. Overnight culture should have been done the day before. Refer to growing an overnight culture.
2. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube.
3. Add 250 μl Buffer P2 and mix throughly by inverting the tube 4-6 times.
4. Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.
5. Centrifuge for 10 minutes at 13,000 rpm in a table-top microcentrifuge.
6. Apply the supernatant to the QIAprep spin column by decanting or pipetting.
7. Centrifuge for 30-60 seconds. Discard the flow-through.
8. Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30-60 seconds. Discard the flow-through.
9. Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30-60 seconds.
10. Discard the flow-through, and centrifuge for an additional 1 minute to remove residual wash buffer.
11. To elute DNA, place the QIAprep column in a clean 1.5 ml microcentrifuge tube. Add 50 μl Buffer EB or water to the center of each QIAprep spin column, let stand for 1 minute, and centrifuge for 1 minute.

Go back to our Protocol List