|QIAprep Spin Miniprep Kit Protocol (using a microcentrifuge)||[back]|
This protocol is designed for purification of up to 20 µg of high-copy plasmid DNA from 1–5 ml overnight cultures of E. coli in LB (Luria-Bertani) medium. For purification of low-copy plasmids and cosmids, large plasmids (>10 kb), and DNA prepared using other methods, refer to the recommendations on page 35.
Please read Important Notes for QIAprep Procedures on pages 18–19
1. Resuspend pelleted bacterial cells in 250 µl Buffer P1 and
transfer to a microcentrifuge tube.
2. Add 250 µl Buffer P2 and gently invert the tube 4–6
times to mix.
3. Add 350 µl Buffer N3 and invert the tube immediately but gently
4. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top
5. Apply the supernatants from step 4 to the QIAprep Spin Column by decanting or pipetting.
6. Centrifuge for 30–60 s. Discard the flow-through.
7. (Optional): Wash the QIAprep Spin Column by adding 0.5 ml Buffer
PB and centrifuging for 30–60 s. Discard the flow-through.
8. Wash QIAprep Spin Column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s.
9. Discard the flow-through, and centrifuge for an additional 1 min
to remove residual wash buffer.
10. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube.
To elute DNA, add 50 µl Buffer EB (10 mM Tris·Cl, pH 8.5)
or water to the center of each QIAprep Spin Column, let stand for 1
min, and centrifuge for 1 min.
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