Protocol: Plasmid or Cosmid DNA Purification Using QIAGEN Plasmid Midi and Maxi Kits

''Procedure''

1. Pick a single colony from a freshly streaked selective plate and inoculate a starter
culture of 2–5 ml LB medium containing the appropriate selective antibiotic. Incubate
for approx. 8 h at 37°C with vigorous shaking (approx. 300 rpm).
Use a tube or flask with a volume of at least 4 times the volume of the culture.

2. Dilute the starter culture 1/500 to 1/1000 into selective LB medium. For high-copy
plasmids, inoculate ▲ 25 ml or ● 100 ml medium with ▲ 25–50 μl or

● 100–200 μl of starter culture. For low-copy plasmids, inoculate ▲ 100 ml or

● 500 ml medium with ▲ 100–200 μl or ● 250–500 μl of starter culture. Grow at
37°C for 12–16 h with vigorous shaking (approx. 300 rpm).

Use a flask or vessel with a volume of at least 4 times the volume of the culture. The
culture should reach a cell density of approximately 3–4 x 109 cells per milliliter,
which typically corresponds to a pellet wet weight of approximately 3 g/liter
medium.

3. Harvest the bacterial cells by centrifugation at 6000 x g for 15 min at 4°C.
ƒ If you wish to stop the protocol and continue later, freeze the cell pellets at –20°C.

4. Resuspend the bacterial pellet in ▲ 4 ml or ● 10 ml Buffer P1.
For efficient lysis it is important to use a vessel that is large enough to allow complete
mixing of the lysis buffers. Ensure that RNase A has been added to Buffer P1.
If LyseBlue reagent has been added to Buffer P1, vigorously shake the buffer bottle
before use to ensure LyseBlue particles are completely resuspended. The bacteria
should be resuspended completely by vortexing or pipetting up and down until no
cell clumps remain.

5. Add ▲ 4 ml or ● 10 ml Buffer P2, mix thoroughly by vigorously inverting the sealed
tube 4–6 times, and incubate at room temperature (15–25°C) for 5 min.
Do not vortex, as this will result in shearing of genomic DNA. The lysate should
appear viscous. Do not allow the lysis reaction to proceed for more than 5 min. After
use, the bottle containing Buffer P2 should be closed immediately to avoid
acidification from CO2 in the air.
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.

6. Add ▲ 4 ml or ● 10 ml of chilled Buffer P3, mix immediately and thoroughly by
vigorously inverting 4–6 times, and incubate on ice for ▲ 15 min or ● 20 min.
Precipitation is enhanced by using chilled Buffer P3 and incubating on ice. After
addition of Buffer P3, a fluffy white material forms and the lysate becomes less
viscous. The precipitated material contains genomic DNA, proteins, cell debris, and
KDS. The lysate should be mixed thoroughly to ensure even potassium dodecyl sulfate
precipitation. If the mixture still appears viscous, more mixing is required to
completely neutralize the solution.
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.

7. Centrifuge at ≥20,000 x g for 30 min at 4°C. Remove supernatant containing plasmid
DNA promptly.
Before loading the centrifuge, the sample should be mixed again. Centrifugation
should be performed in non-glass tubes (e.g., polypropylene). After centrifugation
the supernatant should be clear.
Note: Instead of centrifugation steps 7 and 8, the lysate can be efficiently cleared by
filtration using a QIAfilter Kits or Cartridges (see www.qiagen.com/products/
plasmid/LargeScaleKits).

8. Centrifuge the supernatant again at ≥20,000 x g for 15 min at 4°C. Remove
supernatant containing plasmid DNA promptly.
This second centrifugation step should be carried out to avoid applying suspended
or particulate material to the QIAGEN-tip. Suspended material (causing the sample
to appear turbid) can clog the QIAGEN-tip and reduce or eliminate gravity flow.

☞ Remove a ▲ 240 μl or ● 120 μl sample from the cleared lysate supernatant and
save for an analytical gel (sample 1) in order to determine whether growth and
lysis conditions were optimal.

9. Equilibrate a ▲ QIAGEN-tip 100 or ● QIAGEN-tip 500 by applying ▲ 4 ml or
● 10 ml Buffer QBT, and allow the column to empty by gravity flow.
Flow of buffer will begin automatically by reduction in surface tension due to the
presence of detergent in the equilibration buffer. Allow the QIAGEN-tip to drain
completely. QIAGEN-tips can be left unattended, since the flow of buffer will stop
when the meniscus reaches the upper frit in the column.
10. Apply the supernatant from step 8 to the QIAGEN-tip and allow it to enter the resin
by gravity flow.
The supernatant should be loaded onto the QIAGEN-tip promptly. If it is left too long
and becomes cloudy due to further precipitation of protein, it must be centrifuged
again or filtered before loading to prevent clogging of the QIAGEN-tip.

☞ Remove a ▲ 240 μl or ● 120 μl sample from the flow-through and save for an
analytical gel (sample 2) in order to determine the efficiency of DNA binding to
the QIAGEN Resin.
11. Wash the QIAGEN-tip with ▲ 2 x 10 ml or ● 2 x 30 ml Buffer QC.
Allow Buffer QC to move through the QIAGEN-tip by gravity flow. The first wash is
sufficient to remove all contaminants in the majority of plasmid DNA preparations.
The second wash is especially necessary when large culture volumes or bacterial
strains producing large amounts of carbohydrates are used.

☞ Remove a ▲ 400 μl or ● 240 μl sample from the combined wash fractions and
save for an analytical gel (sample 3).

12. Elute DNA with ▲ 5 ml or ● 15 ml Buffer QF.
Collect the eluate in a 15 ml or 50 ml tube (not supplied). Use of polycarbonate
centrifuge tubes is not recommended as polycarbonate is not resistant to the alcohol
used in subsequent steps.
Note: For constructs larger than 45–50 kb, prewarming the elution buffer to 65°C
may help to increase yield.

☞ Remove a ▲ 100 μl or ● 60 μl sample of the eluate and save for an analytical
gel (sample 4).
ƒ If you wish to stop the protocol and continue later, store the eluate at 4°C. Storage
periods longer than overnight are not recommended.

13. Precipitate DNA by adding ▲ 3.5 ml or ● 10.5 ml (0.7 volumes) room-temperature
isopropanol to the eluted DNA. Mix and centrifuge immediately at ≥15,000 x g for
30 min at 4°C. Carefully decant the supernatant.
All solutions should be at room temperature in order to minimize salt precipitation,
although centrifugation is carried out at 4°C to prevent overheating of the sample.
Alternatively, disposable conical bottom centrifuge tubes can be used for
centrifugation at 5000 x g for 60 min at 4°C. Isopropanol pellets have a glassy
appearance and may be more difficult to see than the fluffy, salt-containing pellets
that result from ethanol precipitation. Marking the outside of the tube before
centrifugation allows the pellet to be more easily located. Isopropanol pellets are also
more loosely attached to the side of the tube, and care should be taken when
removing the supernatant.

14. Wash DNA pellet with ▲ 2 ml or ● 5 ml of room-temperature 70% ethanol, and
centrifuge at ≥15,000 x g for 10 min. Carefully decant the supernatant without
disturbing the pellet.
Alternatively, disposable conical-bottom centrifuge tubes can be used for
centrifugation at 5000 x g for 60 min at 4°C. The 70% ethanol removes precipitated
salt and replaces isopropanol with the more volatile ethanol, making the DNA easier
to redissolve.

15. Air-dry the pellet for 5–10 min, and redissolve the DNA in a suitable volume of buffer
(e.g., TE buffer, pH 8.0, or 10 mM Tris·Cl, pH 8.5).
Redissolve the DNA pellet by rinsing the walls to recover all the DNA, especially if
glass tubes have been used. Pipetting the DNA up and down to promote
resuspension may cause shearing and should be avoided. Overdrying the pellet will
make the DNA difficult to redissolve. DNA dissolves best under slightly alkaline
conditions; it does not easily dissolve in acidic buffers.

''Determination of yield''

To determine the yield, DNA concentration should be determined by both UV
spectrophotometry at 260 nm and quantitative analysis on an agarose gel. For reliable
spectrophotometric DNA quantification, A260 readings should lie between 0.1 and 1.0.

Agarose gel analysis

We recommend removing and saving aliquots during the purification procedure
(samples 1–4). If the plasmid DNA is of low yield or quality, the samples can be
analyzed by agarose gel electrophoresis to determine at what stage of the purification
procedure the problem occurred