Page 3 - Contents
QIAprep Miniprep Handbook 12/2006 3 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 15 Guidelines for QIAvac ...
Page 4 - Kit Contents; QIAprep Spin Miniprep Kit
Kit Contents QIAprep Spin Miniprep Kit (50) (250) Catalog no. 27104 27106 QIAprep Spin Columns 50 250 Buffer P1 20 ml 73 ml Buffer P2 20 ml 73 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 73 µl RNase A † 200 µl 730 µl ...
Page 5 - QIAprep 8 Turbo Miniprep Kit
QIAprep Miniprep Handbook 12/2006 5 QIAprep 8 Turbo Miniprep Kit (10) (50) Catalog no. 27152 27154 TurboFilter ® 8 Strips 10 50 QIAprep 8 Strips 10 50 Buffer P1 40 ml 125 ml Buffer P2 40 ml 125 ml Buffer N3* 60 ml 2 x 125 ml Buffer PB* 100 ml 500 ml Buffer PE (concentrate) 2 x 20 ml 2 x 100 ml Buffe...
Page 6 - QIAprep 96 Turbo Miniprep Kit
6 QIAprep Miniprep Handbook 12/2006 QIAprep 96 Turbo Miniprep Kit (4) (24) Catalog no. 27191 27193 TurboFilter 96 Plates 4 24 QIAprep 96 Plates 4 24 Buffer P1 125 ml 1 x 700 ml, 1 x 125 ml Buffer P2 125 ml 1 x 700 ml, 1 x 125 ml Buffer N3* 2 x 80 ml 1 x 1000 ml, 1 x 80 ml Buffer PB* 500 ml 6 x 500 m...
Page 8 - Technical Assistance
8 QIAprep Miniprep Handbook 12/2006 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 extensivepractical and theoretical expertise in molecular biology and the use of...
Page 9 - Safety Information; Buffer N3; Poison Information Center Mainz, Germany
QIAprep Miniprep Handbook 12/2006 9 Safety Information When working with chemicals, always wear a suitable lab coat, disposable gloves,and protective goggles. For more information, please consult the appropriate materialsafety data sheets (MSDSs). These are available online in convenient and compact...
Page 10 - Introduction
10 QIAprep Miniprep Handbook 12/2006 Introduction The QIAprep Miniprep system provides a fast, simple, and cost-effective plasmidminiprep method for routine molecular biology laboratory applications. QIAprepMiniprep Kits use silica membrane technology to eliminate the cumbersome steps asso-ciated wi...
Page 11 - Applications using QIAprep purified DNA; Principle; Preparation and clearing of bacterial lysate
QIAprep Miniprep Handbook 12/2006 11 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...
Page 12 - endA; Note
12 QIAprep Miniprep Handbook 12/2006 LyseBlue is a color indicator which provides visual identification of optimum buffer mix-ing. This prevents common handling errors that lead to inefficient cell lysis and incom-plete precipitation of SDS, genomic DNA, and cell debris. This makes LyseBlue idealfor...
Page 13 - QIAprep Spin; Elution Volume versus DNA Concentration and Recovery; Incubation Time versus DNA Recovery
QIAprep Miniprep Handbook 12/2006 13 QIAprep Spin Figure 1 10 µg pUC18 DNA was purified using the QIAprep Spin protocol and eluted with the indicated vol- umes of Buffer EB. The standard protocol uses 50 µl Buffer EB for elution, since this combines high yield withhigh concentration. However the yie...
Page 14 - Using LyseBlue reagent
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 containingpBluescript ® . Elutio...
Page 15 - Important Notes; Growth of bacterial cultures in tubes or flasks; g for; Cell Cultivation in a 96-Well Block for QIAprep Turbo 96; modate the 2 ml flat-bottom blocks before starting the centrifuge.
QIAprep Miniprep Handbook 12/2006 15 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 of1–5 ml LB medium containing th...
Page 16 - Buffer notes; Add the provided RNase A solution to Buffer P1, mix, and store at 2–8°C.; Centrifugation notes; Wear safety glasses when working near a manifold under pressure.; empty; QIAprep
16 QIAprep Miniprep Handbook 12/2006 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 anyprecipitate by warmin...
Page 17 - : Store DNA at –20°C when eluted with water, as DNA may degrade in the
QIAprep Miniprep Handbook 12/2006 17 Table 2. Regulation of Vacuum Pressures for QIAprep Multiwell Procedures Procedure Vacuum Module used for Vacuum pressure ‡ manifold checking pressure* mbar mm Hg QIAprep 8 QIAvac 6S QIAprep 8 strip(s) † –100 to –530 –75 to –400 QIAprep 8 Turbo QIAvac 6S QIAprep ...
Page 18 - Multichannel pipet recommendations; cordless multichannel pipet can be purchased with; Pipet tip recommendations; Finntip; Guidelines for QIAvac manifolds
18 QIAprep Miniprep Handbook 12/2006 Multichannel pipet recommendations Many steps of the QIAprep 8 procedure and the QIAprep 8 and 96 Turbo proceduresrequire repeated pipetting, and a reservoir or multichannel pipet can greatly facilitate liquid handling. The Matrix Impact ® cordless multichannel p...
Page 19 - Table 4. Chemical-Resistance Properties of QIAvac Manifolds; QIAvac vacuum manifolds
■ To ensure consistent performance, do not apply silicone or vacuum grease to anypart 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 isapplied to the assembled unit. To maximize gasket lifetime, rinse ...
Page 20 - QIAvac 6S Manifold; QIAvac 96 Manifold
20 QIAprep Miniprep Handbook 12/2006 QIAvac 6S Manifold 10 8 9 5 7 1 2 4 6 3 6 Figure 4 Components of the QIAvac 6S manifold. 1. QIAvac base, which holds a waste tray, a strip holder, or a microtube rack 2. Waste tray 3. QIAvac strip holder to hold 8-well strips 4. QIAvac top plate with slots for 8-...
Page 22 - Please read “Important Notes” on pages 15–21 before starting.; ml) may require inverting up
QIAprep Spin 22 QIAprep Miniprep Handbook 12/2006 Protocol: Plasmid DNA Purification Using the QIAprepSpin Miniprep Kit and a Microcentrifuge This protocol is designed for purification of up to 20 µg of high-copy plasmid DNA from1–5 ml overnight cultures of E. coli in LB (Luria-Bertani) medium. For ...
Page 23 - Steps 7; For washing steps, centrifugation should be performed at 3000 x
QIAprep Spin QIAprep Miniprep Handbook 12/2006 23 5. 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. Dis...
Page 24 - inverting up to 10 times. The solution should become cloudy.
QIAprep Spin 24 QIAprep Miniprep Handbook 12/2006 Protocol: Plasmid DNA Purification Using the QIAprepSpin 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, ...
Page 25 - Other vacuum manifolds
QIAprep Spin QIAprep Miniprep Handbook 12/2006 25 During centrifugation, prepare the vacuum manifold and QIAprep spin columns: QIAvac 24 Plus (see pages 16 and 18–19): ■ Ensure that the vacuum source is connected to the upper threaded hole of theQIAvac 24 Plus and the lower threaded hole is tightly ...
Page 27 - QIAprep 8 Procedure
QIAprep Miniprep Handbook 12/2006 27 QIAprep 8 Procedure Pelleted bacteria ResuspendLyseNeutralize Bind Pure plasmid DNA Elute Wash Vacuum Vacuum Vacuum QIAprep Spin
Page 28 - QIAprep 8; Note: All protocol steps should be carried out at room temperature.; A compact white pellet will form.; During centrifugation, prepare QIAvac 6S:
QIAprep 8 28 QIAprep Miniprep Handbook 12/2006 Protocol: Plasmid DNA Purification Using the QIAprep8 Miniprep Kit This protocol is designed for purification of high-copy plasmid DNA from up to 48 samplesin parallel. Up to 20 µg DNA can be purified from 1–5 ml cultures of E. coli grown in LB (Luria-B...
Page 29 - 1a. For elution into collection microtubes provided:
QIAprep 8 QIAprep Miniprep Handbook 12/2006 29 Make sure the QIAvac 6S is assembled correctly before loading. Load the super-natants 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-t...
Page 30 - QIAprep 8 Turbo; QIAprep 8 and 96 Turbo
QIAprep 8 Turbo 30 QIAprep Miniprep Handbook 12/2006 QIAprep 8 and 96 Turbo Procedure ResuspendLyseNeutralize Pure plasmid DNA Bind Elute Wash Pelleted bacteria Filter QIAprep Vacuum TurboFilter Vacuum Vacuum Vacuum
Page 32 - The flow-through is collected in the waste tray.; 0a. For elution into collection microtubes provided:
QIAprep 8 Turbo 32 QIAprep Miniprep Handbook 12/2006 5. Switch off vacuum, and ventilate the QIAvac 6S slowly. Discard the TurboFilterstrips. Transfer the QIAprep strips containing the cleared lysates into the slots ofthe QIAvac top plate. Close QIAvac 6S lid. Replace strip holder in the base withth...
Page 33 - QIAprep 96 Turbo
QIAprep 96 Turbo QIAprep Miniprep Handbook 12/2006 33 Protocol: Plasmid DNA Purification Using the QIAprep96 Turbo Miniprep Kit This protocol is designed for high-throughput plasmid DNA minipreps using TurboFilter 96and QIAprep 96 plates on QIAvac 96. The kit accommodates up to 96 parallel preparati...
Page 35 - 0a. For elution into provided collection microtubes:
QIAprep 96 Turbo QIAprep Miniprep Handbook 12/2006 35 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 theQIAprep plate is seated securely. ...
Page 36 - Troubleshooting Guide; Comments and suggestions
36 QIAprep Miniprep Handbook 12/2006 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 youmay have about either the information and protocol(s) in this handbook ...
Page 39 - Appendix A: Background Information; Growth of bacterial cultures
QIAprep Miniprep Handbook 12/2006 39 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 maydepend on factors such as plasm...
Page 40 - Host strains; Most; Inoculation; par locus which ensures that the plasmids segregate equally
40 QIAprep Miniprep Handbook 12/2006 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. Hoststrains such as DH1, DH5 α , and C600 give high-quality DNA. The slower growin...
Page 42 - Preparation of cell lysates
42 QIAprep Miniprep Handbook 12/2006 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 cel...
Page 43 - Appendix B: Agarose Gel Analysis of Plasmid DNA; Agarose Gel Analysis
QIAprep Miniprep Handbook 12/2006 43 Appendix B: Agarose Gel Analysis of Plasmid DNA The QIAprep Miniprep procedure can be analyzed using agarose gel electrophoresisas shown in Figure 6. Samples can be taken from the cleared lysate and its flow-through, precipitated with isopropanol and resuspended ...
Page 44 - Appendix C: Special Applications; Purification of low-copy plasmids and cosmids; E. coli cultures grown in LB; Purification of plasmid DNA prepared by other methods; 00 μl Buffer PB to 100 μl of DNA sample).
44 QIAprep Miniprep Handbook 12/2006 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 two s...
Page 45 - References
QIAprep Miniprep Handbook 12/2006 45 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 plasmidDN...
Page 46 - Ordering Information; Product
46 QIAprep Miniprep Handbook 12/2006 Ordering Information Product Contents Cat. no. QIAprep Spin For 50 plasmid minipreps: 27104 Miniprep Kit (50) 50 QIAprep Spin Columns, Reagents, Buffers, Collection Tubes (2 ml) QIAprep Spin For 250 plasmid minipreps: 27106 Miniprep Kit (250) 250 QIAprep Spin Col...
Page 50 - Notes
Page 51 - QIAGEN Distributors and Importers
QIAprep Miniprep Handbook 12/2006 51 QIAGEN Distributors and Importers Please see the back cover for contact information for your local QIAGEN office. Argentina Tecnolab S.A.Tel: (011) 4555 0010 Fax: (011) 4553 3331 E-mail: [email protected] Bosnia-Herzegovina MEDILINE d.o.o.Tel.: +386 1 830-80-4...
Page 53 - Important
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 DNAfrom 1–5 ml overnight E. coli culture in LB medium using a vacuum manifold with luer connectors. New users and users wanting to pu...
