From page 22 of the QIAGEN miniprep handbook..
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 44.
Please read “Important Notes” on pages 15--21 before starting.
http://kirschner.med.harvard.edu/files/protocols/QIAGEN_QIAprepMiniprepKit_EN.pdf
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 microcentrifuge
tube.
Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible
after resuspension of the pellet.
If LyseBlue reagent has been added to Buffer P1, vigorously shake the buffer bottle
to ensure LyseBlue particles are completely dissolved. The bacteria should be
resuspended completely by vortexing or pipetting up and down until no cell clumps
remain.
2. Add 250 μl Buffer P2 and mix thoroughly by 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 the lysis reaction to proceed for more than
5 min.
If LyseBlue has been added to Buffer P1 the cell suspension will turn blue after addition
of Buffer P2. Mixing should result in a homogeneously colored suspension. If
the suspension contains localized colorless regions or if brownish cell clumps are
still visible, continue mixing the solution until a homogeneously colored suspension
is achieved.
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 thoroughly, immediately after
addition of Buffer N3. Large culture volumes (e.g. ≥5 ml) may require inverting up
to 10 times. The solution should become cloudy.
If LyseBlue reagent has been used, the suspension should be mixed until all trace
of blue has gone and the suspension is colorless. A homogeneous colorless suspension
indicates that the SDS has been effectively precipitated.
4. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
A compact white pellet will form.