Troubleshooting guide
QIAprep 8
28 QIAprep Miniprep Handbook 12/2006
Protocol: Plasmid DNA Purification Using the QIAprep
8 Miniprep Kit
This protocol is designed for purification of high-copy plasmid DNA from up to 48 samples
in parallel. Up to 20 µg DNA can be purified from 1–5 ml cultures of
E. coli
grown 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 recommend-
ations on page 44.
Please read “Important Notes for QIAprep Procedures” on pages 15–21 before starting.
Note: All protocol steps should be carried out at room temperature.
Procedure
1. Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a micro-
centrifuge tube.
Ensure that RNase A has been added to Buffer P1. No cell clumps should be
visible after resuspension of the pellet.
2. Add 250 µl Buffer P2 and mix thoroughly by gently inverting the tube 4–6 times.
Mix gently by inverting the tube. Do not vortex, as this will result in shearing of
genomic DNA. If necessary, continue inverting the tube until the solution becomes
viscous and slightly clear. Do not allow lysis reaction to proceed for more than
5 min.
3. Add 350 µl Buffer N3 and mix immediately and thoroughly by inverting the tube
4–6 times.
To avoid localized precipitation, mix the solution gently but thoroughly, immedi-
ately after addition of Buffer N3. The solution should become cloudy.
4. Centrifuge for 10 min at 13,000 rpm (~17,900 x
g
) in a table-top microcentrifuge.
A compact white pellet will form.
During centrifugation, prepare QIAvac 6S:
(Note: The following procedure applies to the manifold with a hinged lid and
spring lock. See pages 16 and 18–20).
■ Open the QIAvac 6S lid and place QIAprep 8 strips in the slots of the QIAvac
top plate, making sure the strips are positioned tightly. Seal any unused slots
with blanks provided with the QIAvac 6S, and close the QIAvac 6S lid.
■ Place the waste tray inside the QIAvac base. Place the top plate squarely over
the base. Seal any unused wells of the QIAprep strips with strip caps provided.
Attach the QIAvac 6S to a vacuum source.
5. Apply the supernatants from step 4 to the wells of the QIAprep 8 strips and switch
on vacuum source.