HB-1206-006 HB QIAprep Miniprep 0320 WW PDF

March 2020
QIAprep® Miniprep
Handbook
For purification of molecular biology–grade DNA
Plasmids
Large plasmids (>10 kb)
Low-copy plasmids and cosmids
Plasmid DNA prepared by other methods
Sample to Insight__
Contents
Kit Contents................................................................................................................ 4
Storage ..................................................................................................................... 6
Intended Use .............................................................................................................. 6
Safety Information ....................................................................................................... 7
Quality Control ........................................................................................................... 7
Introduction ................................................................................................................ 8
Low throughput................................................................................................. 8
High throughput ............................................................................................... 8
Applications using QIAprep-purified DNA ........................................................... 9
Automated purification of DNA on QIAcube Instruments ....................................... 9
Principle ........................................................................................................ 10
Using LyseBlue reagent.................................................................................... 14
Equipment and Reagents to Be Supplied by User .......................................................... 17
Important Notes ........................................................................................................ 18
Guidelines for QIAvac manifolds ...................................................................... 22
QIAprep Spin procedure ................................................................................. 25
Protocol: Plasmid DNA Purification using the QIAprep Spin Miniprep Kit and a
Microcentrifuge .............................................................................................. 26
Protocol: Plasmid DNA Purification using the QIAprep Spin Miniprep Kit and 5 ml
Collection Tubes ............................................................................................. 28
Protocol: Plasmid DNA Purification using the QIAprep Spin Miniprep Kit and a
Vacuum Manifold ........................................................................................... 29
Protocol: Plasmid DNA Purification using the QIAprep 96 Turbo Miniprep Kit .................. 32
2 QIAprep Miniprep Handbook 03/2020

Troubleshooting Guide .............................................................................................. 36
Appendix A: Background Information ......................................................................... 39
Growth of bacterial cultures ............................................................................. 39
Preparation of cell lysates ................................................................................ 42
Appendix B: Agarose Gel Analysis of Plasmid DNA .................................................. 44
Appendix C: Special Applications .............................................................................. 45
Purification of low-copy plasmids and cosmids ................................................... 45
Purification of plasmid DNA prepared by other methods ..................................... 46
Ordering Information ................................................................................................ 47
Document Revision History ......................................................................................... 52
QIAprep Miniprep Handbook 03/2020 3

Kit Contents
QIAprep Spin Miniprep Kit (50) (250)
Catalog no. 27104 27106
QIAprep 2.0 Spin Columns 50 250
Buffer P1 20 ml 1 x 20 ml, 1 x 50 ml
Buffer N3* 30 ml 140 ml
Buffer PB* 30 ml 150 ml
Buffer PE (concentrate) 2 x 6 ml 55 ml
Buffer EB 15 ml 55 ml
LyseBlue ®
20 µl 1 x 20 µl, 1 x 50 µl
Loading dye 110 µl 550 µl
RNase A† 2 mg 1 x 2 mg, 1 x 5 mg
Collection tubes (2 ml) 50 250
Quick-Start Protocol 1 1
* Buffers N3 and PB contain chaotropic salts which are irritants and not compatible with disinfecting agents containing bleach.
Take appropriate laboratory safety measures and wear gloves when handling. See page 7 for safety information.
†
Provided as a 100 mg/ml solution.

QIAprep 96 Turbo Miniprep Kit (4) (24)
Catalog no. 27191 27193
TurboFilter® 96 Plates 4 24
QIAprep 96 Plates 4 24
Buffer N3* 2 x 80 ml 3 x 30 ml, 2 x 500 ml
Buffer PB* 500 ml 6 x 500 ml
Buffer PE (concentrate) 2 x 100 ml 6 x 200 ml
Buffer EB 2 x 55 ml 1 x 55 ml, 2 x 250 ml
RNase A †
1 x 125 µl 1 x 15 mg, 3 x 25 mg
Tape pads 1 6
Rack of collection microtubes (1.2 ml) 4 24
Caps for collection microtubes 55 x 8 6 x 55 x 8
S-Blocks 4 24
Quick-Start Protocol 1 1
* Buffers N3 and PB contain chaotropic salts which are irritants and not compatible with disinfecting agents containing bleach.
Take appropriate laboratory safety measures and wear gloves when handling. See page 7 for safety information.
†
Provided as a 100 mg/ml solution.

Storage
QIAprep Miniprep Kits should be stored dry at room temperature (15–25°C). Under these
conditions, if no expiration date is mentioned on the kit label, QIAprep Miniprep Kits can be
stored for up to 12 months without showing any reduction in performance and quality. For
longer storage, these kits can be kept at 2–8°C. If any precipitate forms in the buffers after
storage at 2–8°C, it should be redissolved by warming the buffers to 37°C before use.
After addition of RNase A and optional LyseBlue reagent, Buffer P1 is stable for 6 months
when stored at 2–8°C. RNase A stock solution can be stored for 2 years at room temperature.
Intended Use
QIAprep Miniprep Kits are intended for molecular biology applications. These products are
not intended for the diagnosis, prevention or treatment of a disease.
QIAcube® Connect is designed to perform fully automated purification of nucleic acids and
proteins in molecular biology applications. The system is intended for use by professional
users trained in molecular biological techniques and the operation of QIAcube Connect.
All due care and attention should be exercised in the handling of the products. We recommend
all users of QIAGEN® products to adhere to the NIH guidelines that have been developed for
recombinant DNA experiments, or to other applicable guidelines.

Safety Information
When working with chemicals, always wear a suitable lab coat, disposable gloves and
protective goggles. For more information, please consult the appropriate safety data sheets
(SDSs). These are available online in convenient and compact PDF format at
www.qiagen.com/safety where you can find, view and print the SDS for each QIAGEN kit
and kit component.
CAUTION
DO NOT add bleach or acidic solutions directly to the
sample-preparation waste.
Buffers N3 and PB contain guanidine hydrochloride, which can form highly reactive
compounds when combined with bleach.
If liquid containing these buffers is spilt, clean with suitable laboratory detergent and water. If
the spilt liquid contains potentially infectious agents, clean the affected area first with
laboratory detergent and water, and then with 1% (v/v) sodium hypochlorite.
Quality Control
In accordance with QIAGEN’s ISO-certified Quality Management System, each lot of the
QIAprep Miniprep Kit is tested against predetermined specifications to ensure consistent
product quality.

Introduction
The QIAprep Miniprep system provides a fast, simple and cost-effective plasmid miniprep
method for routine molecular biology laboratory applications. QIAprep Miniprep Kits use silica
membrane technology to eliminate the cumbersome steps associated with loose resins or
slurries. Plasmid DNA purified with QIAprep Miniprep Kits is immediately ready for use. Phenol
extraction and ethanol precipitation are not required, and high-quality plasmid DNA is eluted
in a small volume of Tris buffer or water. The QIAprep system consists of 2 products with
different handling options to suit every throughput need.
Low throughput
The QIAprep Spin Miniprep Kit is designed for quick and convenient processing of
1–24 samples simultaneously in less than 30 minutes. QIAprep 2.0 spin columns can be used
in a microcentrifuge or on any vacuum manifold with luer connectors (e.g., QIAvac 24 Plus).
High throughput
The QIAprep 96 Turbo Miniprep Kit enables up to 96 minipreps to be performed

simultaneously in less than 45 minutes on the QIAvac 96. For automated high-throughput
plasmid purification the QIAprep 96 Turbo BioRobot® Kit enables up to 96 minipreps to be
processed in 70 minutes.

Applications using QIAprep-purified DNA
Plasmid DNA prepared using the QIAprep system is suitable for a variety of routine
applications, including:
 Restriction enzyme digestion

 Library screening
 In vitro translation
 Sequencing
 Ligation and transformation
 Transfection of robust cells
Automated purification of DNA on QIAcube Instruments
Purification of DNA can be fully automated on QIAcube Connect or the classic QIAcube. The
innovative QIAcube instruments use advanced technology to process QIAGEN spin columns,
enabling seamless integration of automated, low-throughput sample prep into your laboratory
workflow. Sample preparation using QIAcube instruments follows the same steps as the
manual procedure (i.e., lyse, bind, wash and elute), enabling you to continue using the
QIAprep Spin Miniprep Kit for purification of high-quality DNA.
QIAcube instruments are preinstalled with protocols for purification of plasmid DNA, genomic
DNA, RNA, viral nucleic acids and proteins, plus DNA and RNA cleanup. The range of
protocols available is continually expanding, and additional QIAGEN protocols can be
downloaded free of charge at www.qiagen.com/qiacubeprotocols.

QIAcube Connect.
Principle
The QIAprep Miniprep procedure is based on alkaline lysis of bacterial cells followed by
adsorption of DNA onto silica in the presence of high salt (1). The unique silica membrane used
in the QIAprep Miniprep Kit completely replaces glass or silica slurries for plasmid minipreps.
The procedure consists of 3 basic steps:
 Preparation and clearing of a bacterial lysate

 Adsorption of DNA onto the QIAprep membrane
 Washing and elution of plasmid DNA
All steps are performed without the use of phenol, chloroform, CsCl or ethidium bromide, and
without alcohol precipitation.

Preparation and clearing of bacterial lysate
The QIAprep miniprep procedure uses the modified alkaline lysis method of Birnboim and
Doly (2). Bacteria are lysed under alkaline conditions, and the lysate is subsequently
neutralized and adjusted to high-salt binding conditions in one step. After lysate clearing, the
sample is ready for purification on the QIAprep silica membrane. For more details on growth
of bacterial cultures and alkaline lysis, please refer to Appendix A, page 39. In the QIAprep
Spin procedure, lysates are cleared by centrifugation, while the QIAprep 96 Turbo Miniprep
Kit provides TurboFilter strips or plates for lysate clearing by filtration.
LyseBlue reagent*
Use of LyseBlue is optional and not required to successfully perform plasmid preparations. See
“Using LyseBlue reagent” on page 14 for more information.
LyseBlue is a color indicator that provides visual identification of optimum buffer mixing. This
prevents common handling errors that lead to inefficient cell lysis and incomplete precipitation
of SDS, genomic DNA and cell debris. This makes LyseBlue ideal for use by researchers who
have not had much experience with plasmid preparations, as well as experienced scientists
who want to be assured of maximum product yield.
DNA adsorption to the QIAprep membrane
QIAprep 2.0 columns, strips and plates use a silica membrane for selective adsorption of
plasmid DNA in high-salt buffer and elution in low-salt buffer. The optimized buffers in the lysis
procedure, combined with the unique silica membrane, ensure that only DNA will be
* LyseBlue reagent is only supplied with the QIAprep Spin Miniprep Kit since multiwell or automated formats do not
allow visual control of individual samples.

adsorbed, while RNA, cellular proteins and metabolites are not retained on the membrane but
are found in the flow-through.
Washing and elution of plasmid DNA
Endonucleases are efficiently removed by a brief wash step with Buffer PB. This step is essential
when working with endA+ strains, such as the JM series, HB101 and its derivatives, or any
wild-type strain, to ensure that plasmid DNA is not degraded. The Buffer PB wash step is also
necessary when purifying low-copy plasmids, where large culture volumes are used.
Salts are efficiently removed by a brief wash step with Buffer PE. High-quality plasmid DNA is
then eluted from the QIAprep 2.0 column with 50–100 μl of Buffer EB or water. The purified
DNA is ready for immediate use in a range of applications — no need to precipitate,
concentrate or desalt.
Note: Elution efficiency is dependent on pH. The maximum elution efficiency is achieved
between pH 7.0 and 8.5. When using water for elution, make sure that the pH value is within
this range. Store DNA at –30°C to –15°C when eluted with water, because DNA may degrade
in the absence of a buffering agent.
DNA yield
Plasmid yield with the QIAprep miniprep system varies depending on plasmid copy number
per cell (see Appendix A, page 39), the individual insert in a plasmid, factors that affect
growth of the bacterial culture (see page 39), the elution volume (Figure 1), and the elution
incubation time (Figure 2). A 1.5 ml overnight culture can yield 5–15 μg of plasmid DNA
(Table 1, page 14). To obtain the optimum combination of DNA quality, yield and
concentration, we recommend using Luria Bertani (LB) medium for growth of cultures (for
composition see page Table 8, page 42), eluting plasmid DNA in a volume of 50 μl and
performing a short incubation after addition of the elution buffer.

Figure 1. Elution volume versus DNA concentration and recovery. Using the QIAprep Spin protocol, 10 µg pUC18
DNA was purified and eluted with the indicated volumes of Buffer EB. The standard protocol uses 50 µl Buffer EB for
elution, because this combines high yield with high concentration. However the yield can be increased by increasing
the elution volume.
Figure 2. Incubation time versus DNA recovery. Using the QIAprep Spin Miniprep protocol, 10 μg pBluescript® DNA
was purified and eluted after the indicated incubation times with either 50 μl or 100 μl Buffer EB. The graph shows that
an incubation time of 1 min and doubling the elution buffer volume increase yield.

Table 1. Effect of different compositions of growth medium LB on DNA yield
Culture media Yield
LB (containing 10 g/liter NaCl) 11.5 μg
LB (containing 5 g/liter NaCl) 9.5 μg
QIAprep Spin Miniprep Kit was used to purify DNA from 1.5 ml LB overnight cultures of
XL1-Blue containing pBluescript. Elution was performed according to the standard protocol (50
μl Buffer EB and 1 min incubation). Use of the recommended LB composition (with 10 g/liter
NaCl; see also Appendix A, page 39) provides optimal plasmid yield.
Using LyseBlue reagent
Using a simple visual identification system, LyseBlue reagent prevents common handling errors
that lead to inefficient cell lysis and incomplete precipitation of SDS, cell debris and genomic
DNA.
LyseBlue can be added to the resuspension buffer (Buffer P1) bottle before use. Alternatively,
smaller amounts of LyseBlue can be added to aliquots of Buffer P1, enabling single plasmid
preparations incorporating visual lysis control to be performed.
LyseBlue reagent should be added to Buffer P1 at a ratio of 1:1000 to achieve the required
working concentration (e.g., 10 μl LyseBlue into 10 ml Buffer P1). Make sufficient
LyseBlue/Buffer P1 working solution for the number of plasmid preps being performed.
LyseBlue precipitates after addition into Buffer P1. This precipitate will completely dissolve after
addition of Buffer P2. Shake Buffer P1 before use to resuspend LyseBlue particles.

The plasmid preparation procedure is performed as usual. After addition of Buffer P2 to
Buffer P1, the color of the suspension changes to blue. Mixing should result in a
homogeneously colored suspension. If the suspension contains localized regions of colorless
solution, or if brownish cell clumps are still visible, continue mixing the solution until a
homogeneously colored suspension is achieved.
Upon addition of neutralization buffer (Buffer N3), LyseBlue turns colorless. The presence of a
homogeneous solution with no traces of blue indicates that SDS from the lysis buffer has been
effectively precipitated.
Loading Dye
Loading Dye is provided for analysis of plasmid DNA samples using electrophoresis. It contains
3 marker dyes (bromophenol blue, xylene cyanol and orange G) that facilitate estimation of
DNA migration distance and optimization of agarose gel run time. Refer to Table 2 to identify
the dyes according to migration distance and agarose gel percentage and type. Loading Dye
is supplied as a 5x concentrate; thus 1 volume of Loading Dye should be added to 5 volumes
of purified DNA.
Table 2. Migration distance of gel tracking dyes
%TAE (TBE) Xylene cyanol Bromophenol blue Orange G

agarose gel (light blue) (dark blue) (orange)
0.8 5000 bp (3000 bp) 800 bp (400 bp) 150 bp (<100 bp)
1.0 3000 bp (2000 bp) 400 bp (250 bp) <100 bp (<100 bp)
1.5 1800 bp (1100 bp) 250 bp (100 bp) <100 bp (<100 bp)
2.0 1000 bp (600 bp) 200 bp (<100 bp) <100 bp (<100 bp)
2.5 700 bp (400 bp) 100 bp (<50 bp) <50 bp (<50 bp)

Quantification of DNA fragments
DNA fragments can be quantified by running a sample alongside standards containing known
quantities of the same-sized DNA fragment. The amount of sample DNA loaded can be
estimated by visual comparison of the band intensity with that of the standards.
Figure 3. Agarose gel analysis. An unknown amount of a 5.5 kb DNA fragment (U) was run alongside known
quantities (as indicated in ng) of the same DNA fragment. The unknown sample contained 75–100 ng DNA, as
estimated by visual comparison with the standards. M: 1 kb DNA ladder.

Equipment and Reagents to Be Supplied by User
When working with chemicals, always wear a suitable lab coat, disposable gloves and
protective goggles. For more information, consult the appropriate safety data sheets (SDSs),
available from the product supplier.
 Standard microbiological equipment for growing and harvesting bacteria (e.g.,

inoculating loop, culture tubes and flasks, and 37°C shaking incubator)
 Centrifuge with rotor for 96-well blocks. We recommend QIAGEN’s Centrifuge 4-16S
(cat. no. 80510) for room-temperature centrifugation; for refrigerated centrifugation, we
recommend QIAGEN’s Centrifuge 4-16KS (cat. no. 81610).
 96–100% ethanol
 Vacuum pump (e.g., Vacuum Pump [cat. no. 84010])
 QIAvac 96 (cat. no. 19504)
 Elution Microtube Adapter (available from QIAGEN Technical Services) for QIAvac 96
for the elution step using the QIAvac 96. Alternatively, an empty microplate may be used
to adjust the height of the elution microtubes; or the elution step could be performed in a
suitable centrifuge, where no adapter is necessary.
 If the drying step is performed in a suitable centrifuge (optional), additional S-Blocks
(Square-Well Blocks) or elution microtubes are required.
 If the complete procedure is performed using a centrifuge, additional S-Blocks
are required.

Important Notes
Please read the following notes before starting any of the QIAprep procedures.
Growth of bacterial cultures in tubes or flasks

1. Pick a single colony from a freshly streaked selective plate and inoculate a culture of
1–5 ml LB medium containing the appropriate selective antibiotic. Incubate for 12–16 h
at 37°C with vigorous shaking.
Growth for more than 16 h is not recommended, because cells begin to lyse and plasmid
yields may be reduced. Use a tube or flask with a volume of at least 4 times the volume
of the culture.
2. Harvest the bacterial cells by centrifugation at >8000 rpm (6800 x g) in a conventional
table-top microcentrifuge for 3 min at room temperature (15–25°C).
The bacterial cells can also be harvested in 15 ml centrifuge tubes at 5400 x g for
10 min at 4°C. Remove all traces of supernatant by inverting the open centrifuge tube
until all medium has been drained.
Cell Cultivation in a 96-Well Block for QIAprep Turbo 96
1. Fill each well of a 96-well S-Block with 1.3 ml of growth medium containing the
appropriate selective agent. Inoculate each well from a single bacterial colony. Incubate
the cultures for 20–24 h at 37°C with vigorous shaking.
The wells in the block may be protected against spill-over by covering the block with a
plastic lid or adhesive tape. AirPore microporous tape sheets promote gas exchange
during culturing (see “Ordering Information”, page 47). If nonporous tape is used, use a
needle to pierce 2–3 holes in the tape above each well for aeration.

2. Harvest the bacterial cells in the block by centrifugation for 5 min at 2100 x g in a
centrifuge with a rotor for microtiter plates (e.g., QIAGEN Centrifuge 4K15C or
Heraeus® Minifuge GL), preferably at 4–10°C. The block should be covered with
adhesive tape during centrifugation. Remove media by inverting the block.
To remove the media, peel off the tape and quickly invert the block over a waste
container. Tap the inverted block firmly on a paper towel to remove any remaining
droplets of medium.
WARNING: Ensure that the buckets on the rotor have sufficient clearance to
accommodate the 2 ml S-Blocks before starting the centrifuge.
Buffer notes
 Add the provided RNase A solution to Buffer P1 before use. Use 1 vial RNase A
(centrifuge briefly before use) per bottle Buffer P1 for a final concentration of 100 µg/ml.
Mix and store at 2–8°C.
 Add ethanol (96–100%) to Buffer PE before use (see bottle label for volume).
 Check Buffers P2 and N3 before use for salt precipitation. Redissolve any precipitate by
warming to 37°C. Do not shake Buffer P2 vigorously.
 Close the bottle containing Buffer P2 immediately after use to avoid acidification of Buffer
P2 from CO2 in the air.
 Buffers P2, N3 and PB contain irritants. Wear gloves when handling these buffers.
 Optional: Add the provided LyseBlue reagent to Buffer P1 and mix before use. Use 1 vial
LyseBlue reagent per bottle Buffer P1 for a final dilution of 1:1000 (e.g., 10 µl LyseBlue
into 10 ml Buffer P1). LyseBlue provides visual identification of optimum buffer mixing,
thereby preventing the common handling errors that lead to inefficient cell lysis and
incomplete precipitation of SDS, genomic DNA and cell debris. For more details, see
“Using LyseBlue reagent” on page 14.

Centrifugation notes
 All centrifugation steps are carried out at 13,000 rpm (~17,900 x g) in a conventional
table-top microcentrifuge.
Vacuum notes
 Switch off vacuum between steps to ensure that a consistent, even vacuum is applied
during manipulations.
 Wear safety glasses when working near a manifold under pressure.
 For safety reasons, do not use 96-well plates that have been damaged in any way.
 For the QIAprep 96 Turbo miniprep procedure, the negative pressure (vacuum) should be
regulated before beginning the procedure by applying the vacuum to the appropriate
number of empty QIAprep modules (indicated in Table 3) on the QIAvac manifold.
The vacuum pressure is the pressure differential between the inside of the manifold and
the atmosphere (standard atmospheric pressure: 1013 millibar, or 760 mm Hg) and can
be measured using a vacuum regulator (see “Ordering Information”, page 47). Vacuum
recommendations are given in negative units (Table 3) to indicate the required reduction
in pressure with respect to the atmosphere. Table 4 provides pressure conversions to
other units.
 Use of a vacuum pressure lower than recommended may reduce DNA yield and purity.
Table 3. Regulation of vacuum pressures for QIAprep multiwell procedures
Vacuum pressure†
Module used for
Procedure Vacuum manifold checking pressure* mbar mm Hg
QIAprep 96 Turbo QIAvac 96 QIAprep 96 plate –200 to −40 –150 to −30
* Pressure should be regulated using empty modules on the manifold.

†
Values apply to empty modules on QIAvac. During the working procedure the vacuum may exceed the values
indicated.

Table 4. Pressure conversions
To convert from millibars (mbar) to Multiply by
Millimeters of mercury (mm Hg) 0.75
Kilopascals (kPa) 0.1
Inches of mercury (inch Hg) 0.0295
Torrs (Torr) 0.75
Atmospheres (atm) 0.000987
Pounds per square inch (psi) 0.0145
Elution notes
 Ensure that the elution buffer is dispensed directly onto the center of the QIAprep
membrane for optimal elution of DNA. Average eluate volume is 48 μl from an elution
buffer volume of 50 μl (QIAprep spin procedures), and 60 μl from an elution buffer
volume of 100 μl (QIAprep multiwell procedures).
 For increased DNA yield, use a higher elution-buffer volume. For increased DNA
concentration, use a lower elution-buffer volume (see “DNA yield”, page 12).
 If water is used for elution, make sure that its pH is between 7.0 and 8.5. Elution
efficiency is dependent on pH, and the maximum elution efficiency is achieved within this
range. A pH <7.0 can decrease yield.
Note: Store DNA at –30°C to –15°C when eluted with water, as DNA may degrade in
the absence of a buffering agent.
 DNA can also be eluted in TE buffer (10 mM Tris·Cl, 1 mM EDTA, pH 8.0), but the EDTA
may inhibit subsequent enzymatic reactions.
Multichannel pipette recommendations
Many steps of the QIAprep 96 Turbo procedure require repeated pipetting, and a reservoir
or multichannel pipette can greatly facilitate liquid handling. The E1-ClipTip™ electronic
pipettes (cat. no. 4672100BT) can be purchased from Thermo Fisher Scientific:
www.thermofisher.com.

Pipette tip recommendations
Some standard 1 ml pipette tips are not easily accommodated in the S-Blocks that are used in
the QIAprep 96 Turbo Miniprep protocol. When pipetting into S-Blocks, we recommend using
pipette tips with 1.25 ml or 1.5 ml fill volume, such as:
 ClipTip 1250 pipette tips for use with the E1-ClipTip pipet mentioned above. These can
be purchased from the supplier listed above.
 Finntip® Multistepper® pipette tips for use with single-channel pipettes. These are
available from Thermo Fisher Scientific: www.thermofisher.com.
Guidelines for QIAvac manifolds
QIAvac 24 Plus and QIAvac 96 facilitate DNA minipreps by providing a convenient modular
vacuum manifold for use with the QIAprep system. The following recommendations should be
followed when handling QIAvac manifolds:
 QIAvac manifolds operate with a house vacuum or Vacuum Pump (e.g., Vacuum Pump,
cat. no. 84010 [USA and Canada], 84000 [Japan], or 84020 [rest of world]).
 Always store QIAvac manifolds clean and dry. To clean, simply rinse all components
with water and dry with paper towels. Do not air dry, as the screws may rust and need
to be replaced. Do not use abrasives or solvents.
 Always place the QIAvac manifold on a secure bench top or work area. If dropped, the
manifold may crack.
 The components of QIAvac manifolds are not resistant to ethanol, methanol or other
organic solvents (Table 5). Do not bring solvents into contact with the vacuum manifold. If
solvents are spilled on the unit, rinse thoroughly with distilled water. Ensure that no
residual Buffer PE remains in the vacuum manifold.

 To ensure consistent performance, do not apply silicone or vacuum grease to any part of a
QIAvac manifold. The spring lock on the top plate and the self-sealing gasket (QIAvac 96)
provide an airtight seal when vacuum is applied to the assembled unit. To maximize
product life, rinse the gasket free of salts and buffers after each use and dry with paper
towels before storage.
Table 5. Chemical-resistance properties of QIAvac manifolds
Resistant to Not resistant to
Chlorine bleach (12%) Acetic acid* Benzene
Hydrochloric acid Acetone Chloroform
Sodium chloride Chromic acid* Ethers
Sodium hydroxide Phenol Toluene
Urea Concentrated alcohols*
* QIAvac 24 Plus is resistant to these chemicals.
QIAvac vacuum manifolds
Figure 4. Components of the QIAvac 24 Plus manifold.

1. QIAvac 24 Plus vacuum manifold
2. Luer slot closed with luer plug
3. Spin column

Figure 5. Components of the QIAvac 96 manifold.
1. QIAvac base, which holds a waste tray, a plate holder or a microtube rack
2. Waste tray
3. Plate holder (shown with 96-well plate)
4. QIAvac 96 top plate with aperture for 96-well plate
5. Microtube rack
6. 96-well plate (not included with QIAvac 96, included in QIAprep 96 Turbo Miniprep Kits)

QIAprep Spin procedure

Protocol: Plasmid DNA Purification using the
QIAprep Spin Miniprep Kit and a
Microcentrifuge
This protocol is designed for the purification of up to 20 μg of high-copy plasmid DNA from
1–5 ml overnight cultures of E. coli in LB medium. For purification of low-copy plasmids and
cosmids, large plasmids (>10 kb), and DNA prepared using other methods, refer to Appendix
C: Special Applications, page 45.
Please read “Important Notes”, page 17, before starting.
Note: All protocol steps should be carried out at room temperature (15–25°C).
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, because 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. 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.
5. Apply 800 μl of the supernatant from step 4 to the QIAprep 2.0 spin column
by pipetting.
6. Centrifuge for 30–60 s. Discard the flow-through.
7. Recommended: Wash the QIAprep 2.0 spin column by adding 0.5 ml Buffer PB and
centrifuging for 30–60 s. Discard the flow-through.
This step is necessary to remove trace nuclease activity when using endA+ strains, such
as the JM series, HB101 and its derivatives, or any wild-type strain, which have high
levels of nuclease activity or high carbohydrate content. Host strains, such as XL-1 Blue
and DH5®α, do not require this additional wash step.
8. Wash QIAprep 2.0 spin column by adding 0.75 ml Buffer PE and centrifuging for
30–60 s.
9. Discard the flow-through, and centrifuge at full speed for an additional 1 min to remove
residual wash buffer.
Important: Residual wash buffer will not be completely removed unless the flow-through is
discarded before this additional centrifugation. Residual ethanol from Buffer PE may
inhibit subsequent enzymatic reactions.
10. Place the QIAprep 2.0 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 2.0
spin column, let stand for 1 min and centrifuge for 1 min.

QIAprep Spin Miniprep Kit and 5 ml
Collection Tubes
The QIAprep Spin Miniprep procedure can be performed using 5 ml centrifuge tubes (e.g.,
Greiner®, cat. no. 115101 or 115261) as collection tubes to decrease handling. The standard
protocol on pages 26–27 should be followed with the following modifications:
Step 4: Place a QIAprep 2.0 spin column in a 5 ml centrifuge tube instead of a

2 ml collection tube.
Step 6: Centrifuge at 3000 x g for 1 min using a suitable rotor (e.g., Beckman®
GS-6KR centrifuge at ~4000 rpm). (The flow-through does not need to be
discarded.)
Steps 7 and 8: For washing steps, centrifugation should be performed at 3000 x g for 1
min. (The flow-through does not need to be discarded.)
Step 9: Transfer the QIAprep 2.0 spin column to a microcentrifuge tube. Centrifuge
at maximum speed for 1 min. Continue with step 10 of the protocol.

Protocol: Plasmid DNA Purification using
the QIAprep Spin Miniprep Kit and a
Vacuum Manifold
This protocol is designed for purification of up to 20 μg high-copy plasmid DNA from 1–5 ml
overnight cultures of E. coli grown in LB medium, using QIAprep 2.0 spin columns on QIAvac
24 Plus or other vacuum manifolds with luer connectors. For purification of low-copy plasmids
and cosmids, large plasmids (>10 kb) and DNA prepared using other methods, refer to
Appendix C, page 45.
Procedure
1. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge
tube.
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 gently 4–6 times.
Do not vortex, because 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. Mix immediately and thoroughly by inverting the tube 4–6 times.
To avoid localized precipitation, mix the solution gently but 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.
During centrifugation, prepare the vacuum manifold and QIAprep 2.0 spin columns.
For QIAvac 24 Plus (see pages 20, and 22–23):
 Ensure that the vacuum source is connected to the upper threaded hole of the
QIAvac 24 Plus and the lower threaded hole is tightly sealed using the screw cap.
 If using the QIAvac Connecting System, connect the system to the manifold and
vacuum soured as described in the QIAvac 24 Plus Handbook.
 Insert up to 24 spin columns into the luer slots of the QIAvac 24 Plus. Close unused
luer slots with luer plugs.
For other vacuum manifolds: Follow the supplier’s instructions. Insert each QIAprep 2.0
column into a luer connector.

5. Apply 800 μl of the supernatant from step 4 to the QIAprep 2.0 spin column by
pipetting.
6. Switch on vacuum source to draw the solution through the QIAprep 2.0 spin columns,
and then switch off vacuum source.
7. Recommended: Wash the QIAprep 2.0 spin column by adding 0.5 ml Buffer PB. Switch
on vacuum source. After the solution has moved through the column, switch off the
vacuum source.
levels of nuclease activity or high carbohydrate content. Host strains such as, XL-1 Blue
and DH5α, do not require this additional wash step.
8. Wash the QIAprep 2.0 spin column by adding 0.75 ml Buffer PE. Switch on vacuum
source to draw the wash solution through the column, and then switch off vacuum source.
9. Transfer the QIAprep 2.0 spin columns to a microcentrifuge tube. Centrifuge for 1min.
Important: This extra spin is necessary to remove residual Buffer PE. Residual ethanol
from Buffer PE may inhibit subsequent enzymatic reactions.
10. Place the QIAprep 2.0 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 the QIAprep 2.0
spin column, let stand for 1 min and centrifuge for 1 min.

QIAprep 96 Turbo Miniprep Kit
This protocol is designed for high-throughput plasmid DNA minipreps using TurboFilter 96 and
QIAprep 96 plates on QIAvac 96. The kit accommodates up to 96 parallel preparations of
up to 20 µg of high-copy plasmid DNA from 1–5 ml overnight cultures of E. coli grown in LB
medium. If 1.3 ml overnight cultures are used, up to 96 cultures can be grown in an S-Block
(see page 18 for protocol). For purification of low-copy plasmids and cosmids, large plasmids
(>10 kb), and DNA prepared using other methods, refer to the recommendations in Appendix
C, page 45. DNA purification can be automated; please call QIAGEN for more details.
Procedure
1. Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to the S-Block (if cells
were not harvested in this block) provided with the kit.
2. Add 250 µl Buffer P2 to each sample. Dry the top of the S-Block with a paper towel, seal
the block with the tape provided, gently invert the block 4–6 times to mix, and incubate
at room temperature for 5 min.
It is important to mix gently by inverting the block. Do not shake vigorously, as this will
result in shearing of genomic DNA. If necessary, continue inverting the block until the
solution becomes viscous and slightly clear.

During incubation, prepare QIAvac 96 (see pages 20–24):
 Place the TurboFilter 96 plate in the QIAvac top plate, and make sure that the plate
is seated securely. Seal unused wells of the TurboFilter with tape.
 Place the plate holder inside the QIAvac base. Place QIAprep 96 plate into the
plate holder.
 Place QIAvac 96 top plate squarely over base. The QIAprep plate should now be
positioned under the TurboFilter plate. Attach QIAvac to a vacuum source.
3. Remove the tape from the block. Add 350 µl Buffer N3 to each sample, dry the top of
the S-Block with a paper towel, and seal the block with a new tape sheet. Gently invert
the block 4–6 times.
To avoid localized precipitation, mix the samples gently but thoroughly, immediately after
addition of Buffer N3. The solutions should become cloudy.
4. Remove the tape from the block. Pipet the lysates from step 3 (850 µl per well) into the
wells of the TurboFilter plate. Unused wells of the TurboFilter plate should be sealed with
tape. Apply vacuum until all samples have passed through.
The optimal flow rate is approximately 1–2 drops per second, which can be regulated by
using a 3-way valve or vacuum regulator (see “Ordering Information”, page 49)
between the QIAvac and the vacuum source.
5. Switch off vacuum and ventilate the QIAvac 96 slowly. Discard the TurboFilter plate.
Transfer the QIAprep plate containing the cleared lysates to the top plate of the manifold.
Seal any unused wells of the QIAprep plate with tape. Replace plate holder in the base
with waste tray. Place the top plate squarely over the base, making sure that the
QIAprep plate is seated securely. Apply vacuum.
The flow-through is collected in the waste tray.

6. Recommended: Switch off vacuum, and wash QIAprep plate by adding 0.9 ml Buffer PB
to each well and applying vacuum.
levels of nuclease activity or high carbohydrate content. Host strains, such as XL-1 Blue
and DH5α, do not require this additional step.
7. Switch off vacuum. Wash QIAprep plate by adding 0.9 ml of Buffer PE to each well and
applying vacuum. Repeat once.
8. After Buffer PE has been drawn through all wells, apply maximum vacuum for an
additional 10 min to dry the membrane.
Important: This step removes residual Buffer PE from the membrane. The removal is only
effective when maximum vacuum is used (i.e., turn off vacuum regulator or leakage
valves if they are used), allowing maximum airflow to go through the wells.
9. Switch off vacuum, and ventilate the QIAvac 96 slowly. Lift the top plate from the base
(not the QIAprep plate from the top plate), vigorously tap the top plate on a stack of
absorbent paper until no drops come out, and blot the nozzles of the QIAprep plate with
clean absorbent paper. Proceed either to step 10a or 10b, as desired.
This step removes residual Buffer PE, which may be present around the outlet nozzles and
collars of QIAprep plate. Residual ethanol from Buffer PE may inhibit subsequent
enzymatic reactions.
10a. For elution into provided collection microtubes:
Replace waste tray with the blue collection microtube rack containing 1.2 ml collection
microtubes. Place the top plate back on the base, making sure that the QIAprep plate is
seated securely.
10b. For elution into a 96-well microplate:
Replace waste tray with an empty blue collection microtube rack (provided with the
QIAvac 96). Place a 96-well microplate directly on the rack. Place the top plate back on
the base, making sure that the QIAprep plate is positioned securely.

11. To elute DNA, add 100 µl of Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of
each well of the QIAprep plate, let stand for 1 min, and apply maximum vacuum for 5
min. Switch off vacuum and ventilate QIAvac 96 slowly.
For increased DNA concentration, an elution volume of 75 µl can be used.

Troubleshooting Guide
This troubleshooting guide may be helpful in solving any problems that may arise. For
more information, see also the Frequently Asked Questions page at our Technical Support
Center: www.qiagen.com/FAQ/FAQList.aspx. The scientists in QIAGEN Technical
Services are always happy to answer any questions you may have about either the
information and/or protocols in this handbook or sample and assay technologies (for
contact information, visit www.qiagen.com).
Comments and suggestions
Low or no yield
General Low yields may be caused by a number of factors. To find the source of the
problem, analyze fractions saved from each step in the procedure on an
agarose gel (e.g., Figure 6, page 44). A small amount of the cleared lysate
and the entire flow-through can be precipitated by adding 0.7 volumes
isopropanol and centrifuging at maximum speed (13,000 rpm or
~17,000 x g) for 30 minutes. The entire wash flow-through can be
precipitated by adding 0.1 volumes of 3 M sodium acetate, pH 5.0, and 0.7
volumes of isopropanol.
No DNA in the cleared lysate before loading
a) Plasmid did not Read “Growth of bacterial cultures” (page 39) and check that the conditions
propagate for optimal growth were met.
b) Lysate prepared Check storage conditions and age of buffers.

incorrectly
c) Buffer P2 precipitated Redissolve by warming to 37°C.
d) Cell resuspension Pelleted cells should be completely resuspended in Buffer P1. Do not add
incomplete Buffer P2 until an even suspension is obtained.
DNA is found in the flow-through of cleared lysate
a) QIAprep membrane If rich culture media, such as TB or 2x YT, are used, culture volumes must
overloaded be reduced. It may be necessary to adjust LB culture volume if the plasmid
and host strain show extremely high copy number or growth rates. See
“Culture media” on page 41.

b) RNase A digestion Ensure that RNase A is added to Buffer P1 before use.

omitted
c) RNase A digestion Reduce culture volume if necessary. If Buffer P1 containing RNase A is

insufficient more than 6 months old, add additional RNase A.
DNA is found in the wash flow-through
Ethanol omitted from Repeat procedure with correctly prepared wash buffer (Buffer PE).
wash buffer
Little or no DNA in eluate
a) Elution buffer incorrect DNA is eluted only in the presence of low-salt buffer (e.g., Buffer EB [10 mM
Tris·Cl, pH 8.5]) or water. Elution efficiency is dependent on pH. The
maximum efficiency is achieved between pH 7.0 and 8.5. When using water
for elution, make sure that the pH value is within this range.
b) Elution buffer incorrectly Add elution buffer to the center of the QIAprep membrane to ensure that
dispensed onto the buffer completely covers the surface of the membrane for maximum
membrane elution efficiency.
Low DNA quality
DNA does not perform well in downstream applications
a) Eluate salt concentration For the QIAprep 2.0 spin column, modify the wash step by adding 0.75 ml
of Buffer PE into the column, centrifuging, and then incubating the column for
5 min at room temperature (15–25°C). For QIAprep 96 Turbo preparations,
ensure that 2 wash steps are carried out prior to elution.
b) Nuclease contamination When using endA+ host strains – such as HB101 and its derivatives, the JM
series, or any wild-type strain – ensure that the wash step with Buffer PB is
performed.
c) Eluate contains Ensure that step 9 in the QIAprep Spin Miniprep protocol (page 27) and
residual ethanol step 9 (page 34) in the QIAprep 96 Turbo Miniprep protocol are performed.
RNA in the eluate
a) RNase A digestion Ensure that RNase A is added to Buffer P1 before use.

omitted
b) RNase A digestion Reduce culture volume if necessary. If Buffer P1 containing RNase A is more
insufficient than 6 months old, add additional RNase A.

Genomic DNA in the eluate
a) Buffer P2 added The lysate must be handled gently after addition of Buffer P2 to prevent
incorrectly shearing. Reduce culture volume if lysate is too viscous for gentle mixing.
b) Buffer N3 added Upon addition of Buffer N3 in step 3 (page 27), mix immediately but gently.
incorrectly
c) Lysis too long Lysis in step 2 (page 26) must not exceed 5 minutes.
d) Culture overgrown Overgrown cultures contain lysed cells and degraded DNA. Do not grow
cultures for longer than 12–16 hours.

Appendix A: Background Information
Growth of bacterial cultures
Plasmids are generally prepared from bacterial cultures grown in the presence of a selective
agent, such as an antibiotic (3, 4). The yield and quality of plasmid DNA may depend on factors,
such as plasmid copy number, host strain, inoculation, antibiotic and type of culture medium.
Plasmid copy number

Plasmids vary widely in their copy number per cell (Table 6), depending on their origin of
replication (e.g., pMB1, ColE1 or pSC101), which determines whether they are under relaxed
or stringent control; and depending on the size of the plasmid and its associated insert. Some
plasmids, such as the pUC series and derivatives, have mutations that allow them to reach
very high copy numbers within the bacterial cell. Plasmids based on pBR322 and cosmids are
generally present in lower copy numbers. Very large plasmids and cosmids are often
maintained at very low copy numbers per cell.
Table 6. Origins of replication and copy numbers of various plasmids (3)
DNA construct Origin of replication Copy number Classification

Plasmids
pUC vectors pMB1* 500–700 High copy
pBluescript vectors ColE1 300–500 High copy
pGEM® vectors pMB1* 300–400 High copy
pTZ vectors pMB1* >1000 High copy
pBR322 and derivatives pMB1* 15–20 Low copy
pACYC and derivatives p15A 10–12 Low copy
pSC101 and derivatives pSC101 ~5 Very low copy
Cosmids
SuperCos ColE1 10–20 Low copy
pWE15 ColE1 10–20 Low copy
* The pMB1 origin of replication is closely related to that of ColE1 and falls in the same incompatibility group. The
high-copy-number plasmids listed here contain mutated versions of this origin.

Host strains
Most E. coli strains can be used successfully to isolate plasmid DNA, although the strain used
to propagate a plasmid has an effect on the quality of the purified DNA. Host strains, such as
DH1, DH5α and C600, give high-quality DNA. The slower-growing strain XL1-Blue also yields
DNA of very high quality, which works extremely well for sequencing. Strain HB101 and its
derivatives, such as TG1 and the JM series, produce large amounts of carbohydrates, which
are released during lysis and can inhibit enzyme activities if not completely removed (4). In
addition, these strains have high levels of endonuclease activity which can reduce DNA
quality. The methylation and growth characteristics of the strain should also be taken into
account when selecting a host strain. XL1-Blue and DH5α are highly recommended for
reproducible and reliable results.
Inoculation
Bacterial cultures for plasmid preparation should always be grown from a single colony picked
from a freshly streaked selective plate. Subculturing directly from glycerol stocks, agar stabs
and liquid cultures may lead to uneven plasmid yield or loss of the plasmid. Inoculation from
plates that have been stored for a long time may also lead to loss or mutation of the plasmid.
The desired clone should be streaked from a glycerol stock onto a freshly prepared agar plate
containing the appropriate selective agent so that single colonies can be isolated. This
procedure should then be repeated to ensure that a single colony of an antibiotic-resistant
clone can be picked. A single colony should be inoculated into 1–5 ml of media containing
the appropriate selective agent and grown with vigorous shaking for 12–16 hours. Growth
for more than 16 hours is not recommended, because cells begin to lyse and plasmid yields
may be reduced.

Antibiotics
Antibiotic selection should be applied at all stages of growth. Many plasmids in use today do
not contain the par locus that ensures that the plasmids segregate equally during cell division.
Daughter cells that do not receive plasmids will replicate much faster than plasmid-containing
cells in the absence of selective pressure, and can quickly take over the culture.
The stability of the selective agent should also be taken into account. Resistance to ampicillin,
for example, is mediated by β-lactamase, which is encoded by the plasmid-linked bla gene
and which hydrolyzes ampicillin. Levels of ampicillin in the culture medium are thus continually
depleted. This phenomenon is clearly demonstrated on ampicillin plates, where “satellite
colonies” appear as the ampicillin is hydrolyzed in the vicinity of a growing colony. Ampicillin
is also very sensitive to temperature, and when in solution should be stored frozen in single-
use aliquots. The recommendations given in Table 7 are based on these considerations.
Table 7. Concentrations of commonly used antibiotics
Stock solutions
Working concentration
Antibiotic Concentration Storage (dilution
Ampicillin (sodium salt) 50 mg/ml in water –30°C to –15°C 100 µg/ml (1/500)
Chloramphenicol 34 mg/ml in ethanol –30°C to –15°C 170 µg/ml (1/200)
Kanamycin 10 mg/ml in water –30°C to –15°C 50 µg/ml (1/200)
Streptomycin 10 mg/ml in water –30°C to –15°C 50 µg/ml (1/200)
Tetracycline HCl 5 mg/ml in ethanol –30°C to –15°C 50 µg/ml (1/100)
Culture media
LB broth is the recommended culture medium for use with QIAprep Kits, because richer broths,
such as TB (Terrific Broth) or 2x YT, lead to extremely high cell densities, which can overload
the purification system. It should be noted that cultures grown in TB may yield 2–5 times the
number of cells compared to cultures grown in LB broth. If these media are used, recommended

culture volumes must be reduced to match the capacity of the QIAprep membrane. If excess
culture volume is used, alkaline lysis will be inefficient, the QIAprep membrane will be
overloaded, and the performance of the system will be unsatisfactory. Furthermore, the
excessive viscosity of the lysate will require vigorous mixing, which may result in shearing of
bacterial genomic DNA and contamination of the plasmid DNA. Care must also be taken if
strains are used which grow unusually fast or to very high cell densities. In such cases, doubling
the volumes of Buffers P1, P2 and N3 may be beneficial. It is best to calculate culture cell
density and adjust the volume accordingly.
Please note that a number of slightly different LB culture broths containing different
concentrations of NaCl are in common use. Although different LB broths produce similar cell
densities after overnight culture, plasmid yields can vary significantly.
Table 8. Recommended composition of Luria Bertani medium
Contents Per liter
Tryptone 10 g
Yeast extract 5g
NaCl 10 g
Preparation of cell lysates
Bacteria are lysed under alkaline conditions. After harvesting and resuspension, the bacterial
cells are lysed in NaOH/SDS (Buffer P2) in the presence of RNase A (2, 5). SDS solubilizes
the phospholipid and protein components of the cell membrane, leading to lysis and release
of the cell contents, while the alkaline conditions denature the chromosomal and plasmid
DNAs, as well as proteins. The optimized lysis time allows maximum release of plasmid DNA
without release of chromosomal DNA, while minimizing the exposure of the plasmid to
denaturing conditions. Long exposure to alkaline conditions may cause the plasmid to become

irreversibly denatured (2). This denatured form of the plasmid runs faster on agarose gels and
is resistant to restriction enzyme digestion.
The lysate is neutralized and adjusted to high-salt–binding conditions in one step by the
addition of Buffer N3. The high salt concentration causes denatured proteins, chromosomal
DNA, cellular debris and SDS to precipitate, while the smaller plasmid DNA renatures correctly
and stays in solution. It is important that the solution is thoroughly and gently mixed to ensure
complete precipitation.
To prevent contamination of plasmid DNA with chromosomal DNA, vigorous stirring and
vortexing must be avoided during lysis. Separation of plasmid from chromosomal DNA is
based on coprecipitation of the cell-wall–bound chromosomal DNA with insoluble complexes
containing salt, detergent and protein. Plasmid DNA remains in the clear supernatant.
Vigorous treatment during the lysis procedure will shear the bacterial chromosome, leaving
free chromosomal DNA fragments in the supernatant. Since chromosomal fragments are
chemically indistinguishable from plasmid DNA under the conditions used, the 2 species will
not be separated on QIAprep membrane and will elute under the same low-salt conditions.
Mixing during the lysis procedure must therefore be carried out by slow, gentle inversion of
the tube.

Appendix B: Agarose Gel Analysis of
Plasmid DNA
The QIAprep Miniprep procedure can be analyzed using agarose gel electrophoresis as
shown in Figure 6. Samples can be taken from the cleared lysate and its flow-through,
precipitated with isopropanol and resuspended in a minimal volume of TE buffer. In Figure 6,
the cleared lysate shows closed circular plasmid DNA and degraded RNase A–resistant RNA.
The flow-through contains only degraded RNA, and no plasmid DNA is present. The eluted
pure plasmid DNA shows no contamination with other nucleic acids.
Figure 6. Agarose gel analysis of the QIAprep Miniprep procedure. C: cleared lysate; F: flow-through;
E: eluted plasmid; M: markers.

Appendix C: Special Applications
Purification of low-copy plasmids and cosmids
All QIAprep miniprep protocols in this handbook can be used for preparation of low-copy-
number plasmid or cosmids from 1–10 ml overnight E. coli cultures grown in LB medium.
Only 2 slight modifications to the protocols are required:
 The wash step with Buffer PB is required for all strains.

 When plasmid or cosmids are >10 kb, preheat Buffer EB (or water) to 70°C prior to
eluting DNA from the QIAprep membrane. A 10 ml overnight LB culture typically yields
5–10 µg DNA.
Note: When using 10 ml culture volume, it is recommended to double the volumes of Buffers
P1, P2 and N3 used.
Purification of very large plasmids (>50 kb)
Plasmids that are >50 kb in size elute less efficiently from silica than smaller plasmids, but do
elute efficiently from the QIAGEN anion-exchange resin. QIAGEN provides the anion-
exchange–based QIAGEN Large-Construct Kit for efficient large-scale purification of ultrapure
genomic DNA-free BAC, PAC, P1 or cosmid DNA. For high-throughput, small-scale purification
of BACs, PACs and P1s, an optimized alkaline lysis protocol in R.E.A.L.® Prep 96 Kits yields
DNA suitable for sequencing and screening. Call QIAGEN Technical Services or your local
distributor for more information on these kits, or see “Ordering Information” on page 47.

Purification of plasmid DNA prepared by other methods
Plasmid DNA isolated by other methods can be further purified using QIAprep modules and
any of the QIAprep protocols in this handbook.
1. Add 5 volumes of Buffer PB to 1 volume of the DNA solution and mix (e.g., add 500 µl
Buffer PB to 100 µl of DNA sample).
2. Apply the samples to QIAprep 2.0 spin columns or to the wells of a QIAprep 96-well
plate. Draw the samples through the QIAprep membrane by centrifugation or vacuum,
and continue the appropriate protocol at the Buffer PE wash step. The optional wash step
with Buffer PB is not necessary.

Ordering Information
Product Contents Cat. no.
QIAprep Spin Miniprep For 50 plasmid minipreps: 27104

Kit (50) 50 QIAprep 2.0 Spin Columns, reagents,
buffers, collection tubes (2 ml), loading dye
QIAprep Spin Miniprep For 250 plasmid minipreps: 27106

Kit (250) 250 QIAprep 2.0 Spin Columns, reagents,
buffers, collection tubes (2 ml), loading dye
QIAprep 2.0 Spin For high-purity plasmid minipreps 27115

Miniprep Columns (100)
QIAprep 96 Turbo For 4 x 96 plasmid minipreps: 27191

Miniprep Kit (4)* 4 TurboFilter 96 Plates, 4 QIAprep 96 Plates,
4 S-Blocks, reagents, buffers, collection
microtubes (1.2 ml), caps
QIAprep 96 Turbo For 24 x 96 plasmid minipreps: 27193

Miniprep Kit (24) 24 TurboFilter 96 Plates, 24 QIAprep 96
Plates, 24 S-Blocks, reagents, buffers,
collection microtubes (1.2 ml), caps
QIAprep 96 Turbo For 4 x 96 plasmid minipreps, 962141

BioRobot Kit (4) 4 each: TurboFilter 96 and QIAprep 96
Plates, S-Blocks, reagents, buffers, collection
microtubes (1.2 ml), caps, 96-well
microplates RB and lids, tape pads
* Requires the use of QIAvac 96.

QIAGEN Plasmid Plus 96 Miniprep and BioRobot Kits — for purification of

transfection grade plasmid DNA in 96-well format
QIAGEN Plasmid Plus 96 For 4 x 96 plasmid minipreps: TurboFilter 96 960241

BioRobot Kit (4) Plates and Plasmid Plus 96 Plates, buffers,
reagents, S-Blocks, and elution microtubes;
for use with the BioRobot Universal System
QIAGEN Plasmid Plus 96 For 4 x 96 plasmid minipreps: TurboFilter 96 16181

Miniprep Kit (4) Plates, Plasmid Plus 96 Plates, buffers,
reagents, S-Blocks, and elution microtubes;
requires use of QIAvac 96 and Elution
Microtube Adapter,* or a centrifugation
system suitable for 96-well blocks
DirectPrep® 96 Kits — for high-throughput plasmid DNA purification
DirectPrep 96 Miniprep For 4 x 96 plasmid minipreps: 27361

Kit (4) †
4 DirectPrep 96 Plates, reagents, buffers,
S-Blocks, AirPore tape sheets, tape pads,
elution microtubes RS, caps
DirectPrep 96 BioRobot For 4 x 96 plasmid minipreps: 962341
Kit (4) ‡
4 DirectPrep 96 Plates, reagents, buffers,
S-Blocks, 96-well microplates RB, AirPore
tape sheets, tape pads
* Requires the use of QIAvac 96.

†
Requires use of QIAvac Multiwell. Larger kit sizes available, please inquire.
‡
For use with BioRobot 3000 or 8000 workstations. Larger kit sizes available, please inquire.

Related products for BAC/PAC/P1 purification
QIAGEN Large-Construct 10 QIAGEN-tip 500, reagents, buffers, 12462

Kit (10) ATP-dependent exonuclease*
QIAvac and accessories
QIAvac 24 Plus Vacuum manifold for processing 1–24 spin 19413

columns: includes QIAvac 24 Plus Vacuum
Manifold, luer plugs, quick couplings
QIAvac 96 Vacuum manifold for processing QIAGEN 19504

96-well plates: includes QIAvac 96 Top
Plate, Base, Waste Tray, Plate Holder
Rack of Collection Microtubes (1.2 ml)
QIAvac Luer Adapter Set † For processing 1–24 QIAprep 2.0 19541
Spin Columns: 6 adapters,
each with 4 luer connectors, 24 plugs
Vacuum Regulator For use with QIAvac manifolds 19530
Vacuum Pump Universal vacuum pump 84000

(100 V, 50/60 Hz) ‡ (capacity 34 L/min, 8 mbar vacuum abs.)

(115 V, 60 Hz) § (capacity 34 L/min, 8 mbar vacuum abs.)
* ATP solution required for exonuclease digestion is not provided.

†
Compatible only with QIAvac Top Plates containing flip-up lid.
‡
Japan.
§
North America.


(230 V, 50 Hz)* (capacity 34 L/min, 8 mbar vacuum abs.)
Automated low-throughput plasmid purification
QIAcube Connect † Instrument, connectivity package, 1-year Inquire

warranty on parts and labor
Starter Pack, QIAcube Filter-tips, 200 µl (1024), 1000 µl filter-tips 990395

(1024), 30 ml reagent bottles (12), rotor
adapters (240), elution tubes (240), rotor
adapter holder
Individual buffers and accessories
Buffer N3 500 ml Buffer N3 19064
Buffer PB 500 ml Buffer PB 19066
Buffer PE (concentrate) 100 ml Buffer PE (concentrate) 19065
RNase A 2.5 ml (100 mg/ml; 7000 units/ml solution) 19101
Collection tubes (2 ml) 1000 collection tubes (2 ml) 19201
Collection microtubes Nonsterile polypropylene tubes (1.2 ml), 19560

(racked) 960 in racks of 96
Collection microtube caps Nonsterile polypropylene caps for collection 19566

microtubes (1.2 ml), 960 in strips of 8, loose
in bag
* Rest of world.
†
All QIAcube Connect instruments are provided with a region-specific connectivity package, including tablet and
equipment necessary to connect to the local network. Further, QIAGEN offers comprehensive instrument service
products, including service agreements, installation, introductory training and preventive subscription. Contact your
local sales representative to learn about your options.

S-Blocks (24) 96-well blocks with 2 ml wells, 24 blocks per 19585

case
Tape Pads (5) Adhesive tape sheets for sealing multiwell 19570
plates and blocks: 25 sheets per pad, 5 pads
per pack
AirPore Tape Sheets (50) Microporous tape sheets for covering 96-well 19571
blocks during bacterial cultivation: 50 sheets
per pack
For up-to-date licensing information and product-specific disclaimers, see the respective
QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are
available at www.qiagen.com or can be requested from QIAGEN Technical Services or your
local distributor.

Document Revision History
Date Changes
March 2019 Changed “flat-bottom blocks” to “S-Blocks”. Updated pipette and pipette tip
recommendations. Transferred Figure 6 into Appendix B, out of Appendix C. Updated
“QIAcube” recommendations and links to “QIAcube Connect”. Added “Equipment and
Reagents to Be Supplied by User” section.
January 2020 Updated text, ordering information and intended use for QIAcube Connect.
March 2020 Deleted loading dye erroneously listed in “Kit Contents” of the QIAprep 96 Turbo
Miniprep Kit.

Limited License Agreement for QIAprep Miniprep Kits
Use of this product signifies the agreement of any purchaser or user of the product to the following terms:
1. The product may be used solely in accordance with the protocols provided with the product and this handbook and for use with components contained in the kit
only. QIAGEN grants no license under any of its intellectual property to use or incorporate the enclosed components of this kit with any components not included
within this kit except as described in the protocols provided with the product, this handbook, and additional protocols available at www.qiagen.com. Some of
these additional protocols have been provided by QIAGEN users for QIAGEN users. These protocols have not been thoroughly tested or optimized by QIAGEN.
QIAGEN neither guarantees them nor warrants that they do not infringe the rights of third-parties.
2. Other than expressly stated licenses, QIAGEN makes no warranty that this kit and/or its use(s) do not infringe the rights of third-parties.
3. This kit and its components are licensed for one-time use and may not be reused, refurbished, or resold.
4. QIAGEN specifically disclaims any other licenses, expressed or implied other than those expressly stated.
5. The purchaser and user of the kit agree not to take or permit anyone else to take any steps that could lead to or facilitate any acts prohibited above. QIAGEN may
enforce the prohibitions of this Limited License Agreement in any Court, and shall recover all its investigative and Court costs, including attorney fees, in any action
to enforce this Limited License Agreement or any of its intellectual property rights relating to the kit and/or its components.
For updated license terms, see www.qiagen.com.
Trademarks: QIAGEN®, Sample to Insight®, QIAcube®, QIAprep®, BioRobot®, DirectPrep®, LyseBlue®, R.E.A.L.®, TurboFilter® (QIAGEN Group); DH5®, E1-ClipTip™,
Finntip®, Multistepper® (Thermo Fisher Scientific or its subsidiaries); Greiner® (Greiner Bio-One GmbH); Heraeus® (Heraeus Holding GmbH); pBluescript® (Agilent
Technologies, Inc.); pGEM® (Promega Corp.); Beckman® (Beckman Coulter, Inc.). Registered names, trademarks, etc. used in this document, even when not specifically
marked as such, are not to be considered unprotected by law.
03/2020 HB-1206-006 © 2020 QIAGEN, all rights reserved

Notes

Notes

Ordering www.qiagen.com/shop | Technical Support support.qiagen.com | Website www.qiagen.com
HB-1206-006
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March 2020

Intended Use .............................................................................................................. 6

Safety Information ....................................................................................................... 7

Quality Control ........................................................................................................... 7

High throughput ............................................................................................... 8

Applications using QIAprep-purified DNA ........................................................... 9

Automated purification of DNA on QIAcube Instruments ....................................... 9

Using LyseBlue reagent.................................................................................... 14

Equipment and Reagents to Be Supplied by User .......................................................... 17

Important Notes ........................................................................................................ 18

Guidelines for QIAvac manifolds ...................................................................... 22

QIAprep Spin procedure ................................................................................. 25

2 QIAprep Miniprep Handbook 03/2020

Appendix A: Background Information ......................................................................... 39

Growth of bacterial cultures ............................................................................. 39

Preparation of cell lysates ................................................................................ 42

Appendix B: Agarose Gel Analysis of Plasmid DNA .................................................. 44

Appendix C: Special Applications .............................................................................. 45

Purification of low-copy plasmids and cosmids ................................................... 45

Purification of plasmid DNA prepared by other methods ..................................... 46

Ordering Information ................................................................................................ 47

Document Revision History ......................................................................................... 52

QIAprep Miniprep Handbook 03/2020 3

QIAprep 2.0 Spin Columns 50 250

Buffer N3* 30 ml 140 ml

Buffer PB* 30 ml 150 ml

Loading dye 110 µl 550 µl

Collection tubes (2 ml) 50 250

4 QIAprep Miniprep Handbook 03/2020

Buffer P1 125 ml 1 x 150 ml, 3 x 250 ml

Buffer P2 125 ml 1 x 150 ml, 3 x 250 ml

Buffer N3* 2 x 80 ml 3 x 30 ml, 2 x 500 ml

Buffer PB* 500 ml 6 x 500 ml

Buffer PE (concentrate) 2 x 100 ml 6 x 200 ml

Buffer EB 2 x 55 ml 1 x 55 ml, 2 x 250 ml

Rack of collection microtubes (1.2 ml) 4 24

Caps for collection microtubes 55 x 8 6 x 55 x 8

QIAprep Miniprep Handbook 03/2020 5

6 QIAprep Miniprep Handbook 03/2020

QIAprep Miniprep Handbook 03/2020 7

The QIAprep 96 Turbo Miniprep Kit enables up to 96 minipreps to be performed

8 QIAprep Miniprep Handbook 03/2020

 Restriction enzyme digestion

Automated purification of DNA on QIAcube Instruments

QIAprep Miniprep Handbook 03/2020 9

The procedure consists of 3 basic steps:

 Preparation and clearing of a bacterial lysate

10 QIAprep Miniprep Handbook 03/2020

DNA adsorption to the QIAprep membrane

QIAprep Miniprep Handbook 03/2020 11

Washing and elution of plasmid DNA

12 QIAprep Miniprep Handbook 03/2020

QIAprep Miniprep Handbook 03/2020 13

Culture media Yield

LB (containing 10 g/liter NaCl) 11.5 μg

LB (containing 5 g/liter NaCl) 9.5 μg

Using LyseBlue reagent

14 QIAprep Miniprep Handbook 03/2020

Table 2. Migration distance of gel tracking dyes