Second Edition December 2006 QIAprep® Miniprep Handbook For purification of molecular biology grade DNA Plasmid Large plasmids (>10 kb) Low-copy plasmids and cosmids Plasmid DNA prepared by other methods W W W. Q I A G E N .
Trademarks: QIAGEN®, QIAprep®, BioRobot®, R.E.A.L.®, TurboFilter® (QIAGEN); DH5α™ (Invitrogen); pBluescript® (Stratagene); pGEM® (Promega Corp.); Beckman® (Beckman Instruments, Inc); Finntip®, Multistepper® (Thermo Electron Oy Corporation); Impact® (Matrix Technologies Corporation, USA); Minifuge® (Heraeus Instruments GmbH). © 2002–2006 QIAGEN, all rights reserved.
Contents Kit Contents 4 Storage 7 Quality Control 7 Product Use Limitations 7 Product Warranty and Satisfaction Guarantee 7 Technical Assistance 8 Safety Information 9 Introduction 10 Principle 11 Using LyseBlue reagent 14 Important Notes Guidelines for QIAvac manifolds 15 18 Protocols Plasmid DNA Purification Using the QIAprep Spin Miniprep Kit and a microcentrifuge 22 and 5 ml collection tubes 23 and a vacuum manifold 24 Plasmid DNA Purification Using the QIAprep 8 Miniprep Kit
Kit Contents QIAprep Spin Miniprep Kit Catalog no. (50) (250) 27104 27106 QIAprep Spin Columns 50 Buffer P1 20 ml 73 ml Buffer P2 20 ml 73 ml Buffer N3* 30 ml 140 ml Buffer PB* 250 30 ml 150 ml 2 x 6 ml 55 ml Buffer EB 15 ml 55 ml LyseBlue 20 µl 73 µl 200 µl 730 µl Buffer PE (concentrate) RNase A † Collection Tubes (2 ml) Handbook QIAprep 8 Miniprep Kit Catalog no.
QIAprep 8 Turbo Miniprep Kit Catalog no. (10) (50) 27152 27154 TurboFilter® 8 Strips 10 50 QIAprep 8 Strips 10 50 Buffer P1 40 ml Buffer P2 40 ml 125 ml Buffer N3* 60 ml 2 x 125 ml 100 ml 500 ml 2 x 20 ml 2 x 100 ml Buffer EB 55 ml 2 x 55 ml RNase A 400 µl † Buffer PB* Buffer PE (concentrate) 125 ml 125 µl ‡ Collection Microtubes (1.
QIAprep 96 Turbo Miniprep Kit Catalog no. (4) (24) 27191 27193 TurboFilter 96 Plates 4 24 QIAprep 96 Plates 4 24 125 ml 1 x 700 ml, 1 x 125 ml Buffer P1 Buffer P2 Buffer N3* Buffer PB* Buffer PE (concentrate) 125 ml 1 x 700 ml, 1 x 125 ml 2 x 80 ml 1 x 1000 ml, 1 x 80 ml 500 ml 6 x 500 ml 2 x 100 ml 5 x 200 ml, 2 x 100 ml Buffer EB 2 x 55 ml 1 x 55 ml, 2 x 250 ml RNase A† 1 x 125 µl 1 x 125 µl, 1 x 700 µl Tape Pads 1 6 Rack of Collection Microtubes (1.
Storage QIAprep Miniprep Kits should be stored dry at room temperature (15–25°C). 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.
Technical Assistance At QIAGEN we pride ourselves on the quality and availability of our technical support. Our Technical Service Departments are staffed by experienced scientists with extensive practical and theoretical expertise in molecular biology and the use of QIAGEN products. If you have any questions or experience any difficulties regarding QIAprep Miniprep Kits, or QIAGEN products in general, please do not hesitate to contact us.
Safety Information When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, please consult the appropriate material safety data sheets (MSDSs). These are available online in convenient and compact PDF format at www.qiagen.com/ts/msds.asp where you can find, view, and print the MSDS for each QIAGEN kit and kit component. CAUTION: DO NOT add bleach or acidic solutions directly to the sample-preparation waste.
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.
Applications using QIAprep purified DNA Plasmid DNA prepared using the QIAprep system is suitable for a variety of routine applications including: ■ Restriction enzyme digestion ■ Sequencing ■ Library screening ■ Ligation and transformation ■ In vitro translation ■ Transfection of robust cells 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).
LyseBlue is a color indicator which 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.
Elution Volume versus DNA Concentration and Recovery QIAprep Spin Figure 1 10 µg pUC18 DNA was purified using the QIAprep Spin protocol and eluted with the indicated volumes of Buffer EB. The standard protocol uses 50 µl Buffer EB for elution, since this combines high yield with high concentration. However the yield can be increased by increasing the elution volume.
Table 1. Effect of Different Compositions of Growth Medium LB on DNA Yield Culture media LB (containing 10 g/liter NaCl) LB (containing 5 g/liter NaCl) Yield 11.5 µg 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, also see Appendix A, p.
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 since cells begin to lyse and plasmid yields may be reduced.
Buffer notes ■ Add the provided RNase A solution to Buffer P1, 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.
Table 2. Regulation of Vacuum Pressures for QIAprep Multiwell Procedures Procedure Vacuum manifold Module used for checking pressure* Vacuum pressure‡ mbar mm Hg QIAprep 8 QIAvac 6S QIAprep 8 strip(s)† –100 to –530 –75 to –400 QIAprep 8 Turbo QIAvac 6S QIAprep 8 strip(s) –100 to –530 –75 to –400 QIAprep 96 Turbo QIAvac 96 QIAprep 96 plate –40 to –200 –30 to –150 † * Pressure should be regulated using empty modules on the manifold.
Multichannel pipet recommendations Many steps of the QIAprep 8 procedure and the QIAprep 8 and 96 Turbo procedures require repeated pipetting, and a reservoir or multichannel pipet can greatly facilitate liquid handling. The Matrix Impact® cordless multichannel pipet can be purchased with an optional expandable tip-spacing system for direct liquid transfer from tubes to microtiter plates. These can be purchased from Matrix Technologies Corporation: www.matrixtechcorp.com .
■ 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 6S and QIAvac 96) provide an airtight seal when vacuum is applied to the assembled unit. To maximize gasket lifetime, rinse the gasket free of salts and buffers after each use and dry with paper towels before storage. Table 4.
QIAvac 6S Manifold 9 8 10 6 4 6 5 3 2 7 1 Figure 4 Components of the QIAvac 6S manifold. 1. QIAvac base, which holds a waste tray, a strip holder, or a microtube rack 5. Microtube rack 2. Waste tray 7. Blanks to seal unused slots 3. QIAvac strip holder to hold 8-well strips 8. QIAvac Luer Adapter† 4. QIAvac top plate with slots for 8-well strips or QIAvac Luer Adapters 6. 8-well strip* 9. QIAprep spin column* 10.
QIAprep Spin Procedure in microcentrifuges on vacuum manifolds Pelleted bacteria Resuspend Lyse Neutralize Bind Bind Vacuum Wash Wash Vacuum Elute Pure plasmid DNA QIAprep Miniprep Handbook 12/2006 Elute Pure plasmid DNA 21
QIAprep Spin Protocol: Plasmid DNA Purification Using the QIAprep Spin Miniprep Kit and a Microcentrifuge 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.
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. Recommended: Wash the QIAprep 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.
QIAprep Spin 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 (Luria-Bertani) medium, using QIAprep spin columns on QIAvac 24 Plus, QIAvac 6S, or other vacuum manifolds with luer connectors.
During centrifugation, prepare the vacuum manifold and QIAprep spin columns: QIAvac 24 Plus (see pages 16 and 18–19): 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.
QIAprep Spin 9. Transfer the QIAprep spin columns to a microcentrifuge tube. Centrifuge for 1 min. 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 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 spin column, let stand for 1 min, and centrifuge for 1 min.
QIAprep 8 Procedure Pelleted bacteria QIAprep Spin Resuspend Lyse Neutralize Bind Vacuum Wash Vacuum Elute Vacuum Pure plasmid DNA QIAprep Miniprep Handbook 12/2006 27
Protocol: Plasmid DNA Purification Using the QIAprep 8 Miniprep Kit QIAprep 8 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 recommendations on page 44.
Make sure the QIAvac 6S is assembled correctly before loading. Load the supernatants promptly onto the QIAprep 8 strips. If the supernatants become cloudy, centrifuge again immediately before loading to prevent clogging the QIAprep 8 strips. The flow-through is collected in the waste tray. 6. Recommended: Switch off vacuum and wash QIAprep 8 strips by adding 1 ml Buffer PB to each well and applying vacuum. 7. Switch off vacuum.
QIAprep 8 and 96 Turbo Procedure Pelleted bacteria Resuspend Lyse Neutralize QIAprep 8 Turbo TurboFilter Filter Vacuum QIAprep Bind Vacuum Wash Vacuum Elute Vacuum Pure plasmid DNA 30 QIAprep Miniprep Handbook 12/2006
Protocol: Plasmid DNA Purification Using the QIAprep 8 Turbo Miniprep Kit This protocol is designed for medium-throughput plasmid DNA minipreps using TurboFilter 8 and QIAprep 8 strips on QIAvac 6S. The kit accommodates 8–48 parallel preparations of up to 20 µg of high-copy plasmid DNA from 1–5 ml overnight cultures of E. coli grown in LB (Luria-Bertani) medium.
5. Switch off vacuum, and ventilate the QIAvac 6S slowly. Discard the TurboFilter strips. Transfer the QIAprep strips containing the cleared lysates into the slots of the QIAvac top plate. Close QIAvac 6S lid. Replace strip holder in the base with the waste tray. Place the top plate squarely over the base. Seal any unused wells of the QIAprep strips with the strip caps provided. Apply vacuum. The flow-through is collected in the waste tray. 6.
Protocol: Plasmid DNA Purification Using the 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 (Luria-Bertani) medium. If 1.3 ml overnight cultures are used, up to 96 cultures can be grown in a flat-bottom block (see page 15 for protocol).
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/s, which can be regulated by using a 3-way valve or vacuum regulator (see page 48) between the QIAvac and the vacuum source. 5. Switch off vacuum and ventilate the QIAvac 96 slowly. Discard the TurboFilter plate.
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.
Troubleshooting Guide This troubleshooting guide may be helpful in solving any problems that may arise. The scientists in QIAGEN Technical Services are always happy to answer any questions you may have about either the information and protocol(s) in this handbook or molecular biology applications (see back cover for contact information). Comments and suggestions Low or no yield General Low yields may be caused by a number of factors.
Comments and suggestions b) RNase A digestion omitted Ensure that RNase A is added to Buffer P1 before use. c) RNase A digestion insufficient Reduce culture volume if necessary. If Buffer P1 containing RNase A is more than 6 months old, add additional RNase A. DNA is found in the wash flow-through Ethanol omitted from wash buffer Repeat procedure with correctly prepared wash buffer (Buffer PE).
Comments and suggestions RNA in the eluate a) RNase A digestion omitted Ensure that RNase A is added to Buffer P1 before use. b) RNase A digestion insufficient Reduce culture volume if necessary. If Buffer P1 containing RNase A is more than 6 months old, add additional RNase A. Genomic DNA in the eluate a) Buffer P2 added incorrectly The lysate must be handled gently after addition of Buffer P2 to prevent shearing. Reduce culture volume if lysate is too viscous for gentle mixing.
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 5), depending on their origin of replication (e.g.
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.
Table 6.
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.
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.
Appendix C: Special Applications Purification of low-copy plasmids and cosmids All QIAprep miniprep protocols in this handbook can be used for preparation of lowcopy-number plasmid or cosmids from 1–10 ml overnight E. coli cultures grown in LB medium. Only two 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, pre-heat Buffer EB (or water) to 70°C prior to eluting DNA from the QIAprep membrane.
References 1. Vogelstein, B., and Gillespie, D. (1979) Preparative and analytical purification of DNA from agarose. Proc. Natl. Acad. Sci. USA 76, 615–619. 2. Birnboim, H.C., and Doly, J. (1979) A rapid alkaline lysis procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7, 1513–1522. 3. Sambrook, J. et al., eds. (1989) Molecular cloning: a laboratory manual. 2nd ed., Cold Spring Harbor Laboratory Press. 4. Ausubel, F.M. et al., eds. (1991) Current protocols in molecular biology.
Ordering Information Product Contents Cat. no. QIAprep Spin Miniprep Kit (50) For 50 plasmid minipreps: 50 QIAprep Spin Columns, Reagents, Buffers, Collection Tubes (2 ml) 27104 QIAprep Spin Miniprep Kit (250) For 250 plasmid minipreps: 250 QIAprep Spin Columns, Reagents, Buffers, Collection Tubes (2 ml) 27106 QIAprep 8 Miniprep Kit (50)* For 50 x 8 plasmid minipreps: 50 QIAprep 8 Strips, Reagents, Buffers, Collection Microtubes (1.
Ordering Information Product QIAprep 96 Turbo BioRobot Kit (4) Contents Cat. no. For 4 x 96 plasmid minipreps, 962141 4 each: TurboFilter 96 and QIAprep 96 Plates, Flat-Bottom Blocks and Lids, Reagents, Buffers, Collection Microtubes (1.
Ordering Information Product Contents Cat. no. QIAvac 6S Vacuum manifold for processing 1–6 QIAGEN 8-well strips: includes QIAvac 6S Top Plate with flip-up lid, Base, Waste Tray, Blanks, Strip Holder Rock of Collection Microtubes (1.2 ml) 19503 QIAvac 96 Vacuum manifold for processing QIAGEN 96-well plates: includes QIAvac 96 Top Plate, Base, Waste Tray, Plate Holder Rock of Collection Microtubes (1.
Ordering Information Product Contents Cat. no. 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 19101 250 mg RNase A (70 U/mg; 100 mg/ml) Collection Tubes (2 ml) 1000 collection tubes (2 ml) 19201 Collection Microtubes Nonsterile polypropylene tubes (1.
Notes 50 QIAprep Miniprep Handbook 12/2006
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Please tear off here Bench Protocol: QIAprep Spin Miniprep Kit Using a Vacuum Manifold This protocol is designed for the purification of up to 20 µg high-copy plasmid DNA from 1–5 ml overnight E. coli culture in LB medium using a vacuum manifold with luer connectors. New users and users wanting to purify low-copy plasmids and cosmids, large plasmids (>10 kb), and DNA prepared using other methods should refer to the detailed protocols provided in the QIAprep Miniprep Handbook, 2nd ed.
This protocol is designed for the purification of up to 20 µg high-copy plasmid DNA from 1–5 ml overnight E. coli culture in LB medium. New users and users wanting to purify low-copy plasmids and cosmids, large plasmids (>10 kb), and DNA prepared using other methods should refer to the detailed protocols provided in the QIAprep Miniprep Handbook, 2nd ed. Things to do before starting ■ Add RNase A solution to Buffer P1. ■ Optional: Add LyseBlue reagent to Buffer P1.
