QIAGEN Plasmid Purification Handbook - …

1 QIAGEN Plasmid Purification HandbookQIAGEN Plasmid Midi, Maxi, Mega, and Giga KitsQIAfilter Plasmid Midi, Maxi, Mega, and Giga KitsEndoFree Plasmid Maxi, Mega, and Giga KitsFor Purification of ultrapure Plasmid DNAS econd EditionAugust Plasmid Purification Handbook 08/2003 Trademarks and disclaimersPatented or patent-pending technology and/or registered or registration-pending trademarks of the QIAGEN Group: QIAGEN QIAfilter QIAprecipitator Effectene , EndoFree , HiSpeed , PolyFect , SuperFect .Beckman is a trademark of Beckman Coulter, is a trademark of Life Technologies, is a registered trademark of Stratagene is a registered trademark of Amersham is a registered trademark of Rohm and Haas is a registered trademark of Promega is a registered trademark of Sorvall Products, of QIAGEN products for PCR is accompanied by a limited license to use them in the Polymerase Chain Reaction (PCR) process for research anddevelopment activities in conjunction with a thermal cycler whose use in the automated performance of the PCR process is covered by the up-front licensefee, either by payment to Applied Biosystems or as purchased, an authorized thermal cycler.

2 The PCR process is covered by Patents 4,683,195and 4,683,202 and foreign equivalents owned by Hoffmann-La Roche names, trademarks, etc. used in this document, even when not specifically marked as such, are not to be considered unprotected by law. 2000 2003 QIAGEN , all rights reserved. QIAGEN Plasmid Purification Handbook 08/20033 ContentsKit Contents5 Storage8 Technical Assistance8 Product Warranty and Satisfaction Guarantee8 Product Use Limitations9 Safety Information9 Introduction10 Comparison of QIAGEN large-scale Plasmid Purification options10 Principle and procedure13 Brief considerations for Plasmid /cosmid Purification procedures13 ProtocolsPlasmid or Cosmid DNA Purification Using QIAGEN Plasmid Midi and Maxi Kits16 Plasmid or Cosmid DNA Purification Using QIAGEN Plasmid Mega and Giga Kits21 Plasmid or Cosmid DNA Purification Using QIAfilter Plasmid Midi and Maxi Kits26 Plasmid or Cosmid DNA Purification Using QIAfilter Plasmid Mega and Giga Kits31 Plasmid or Cosmid DNA Purification Using the EndoFree Plasmid Maxi

3 Kit36 Plasmid or Cosmid DNA Purification Using EndoFree Plasmid Mega and Giga Kits41 Very Low-Copy Plasmid /Cosmid Purification Using QIAGEN -tip 100 or QIAGEN -tip 500 47 Troubleshooting Guide52 Appendix A: Agarose Gel Analysis of the Purification Procedure60 Appendix B: Reliability of DNA Quantification by Spectrophotometry62 Appendix C: General Considerations for Optimal Results63 Growth of bacterial cultures63 Key steps in the Plasmid Purification protocols69 Removal of bacterial endotoxins71 Appendix D: Composition of Buffers74 Appendix E: QIAGEN Anion-Exchange Resin76 Appendix F: Purification of Plasmid DNA Prepared by Other Methods794 QIAGEN Plasmid Purification Handbook 08/2003 Appendix G: Removal of Endotoxins from Purified DNA79 Appendix H.

4 Purification of M13 Replicative Form79 References80 Ordering Information81 QIAGEN Distributors87 QIAGEN Plasmid Purification Handbook 08/20035 Kit ContentsQIAGEN Plasmid KitsQIAGENP lasmid KitMidi (25)Midi (100)Maxi (10)Maxi (25)Maxi (100) Catalog 10025100 QIAGEN -tip 500 10254 x 25 Buffer P11 x 110 ml1 x 440 ml1 x 110 ml 1 x 280 ml 4 x 280 mlBuffer P21 x 110 ml1 x 440 ml1 x 110 ml1 x 280 ml4 x 280 mlBuffer P31 x 110 ml1 x 440 ml1 x 110 ml1 x 280 ml4 x 280 mlBuffer QBT1 x 110 ml1 x 440 ml1 x 110 ml1 x 280 ml4 x 280 mlBuffer QC3 x 190 ml3 x 750 ml3 x 240 ml4 x 425 ml16 x 425 mlBuffer QF1 x 170 ml2 x 280 ml1 x 170 ml1 x 420 ml4 x 420 mlRNase A*1 x 11 mg 1 x 44 mg 1 x 11 mg 1 x 28 mg 4 x 28 mgHandbook 11111 QIAGEN PlasmidPlasmid KitMega (5)Mega (25)Giga (5)

5 Buffer SetCatalog 2500525 QIAGEN -tip10000 5 Buffer P12 x 140 ml2 x 700 ml1 x 700 ml1 x 110 mlBuffer P22 x 140 ml2 x 700 ml1 x 700 ml1 x 110 mlBuffer P32 x 140 ml2 x 700 ml1 x 700 ml1 x 110 mlBuffer QBT1 x 200 ml2 x 500 ml1 x 400 ml1 x 110 mlBuffer QC5 x 220 ml5 x 1000 ml,3 x 1000 ml,6 x 120 ml1 x 500 ml1 x 500 mlBuffer QF1 x 200 ml2 x 510 ml1 x 510 ml2 x 85 mlRNase A*2 x 14 mg 2 x 70 mg 1 x 70 mg1 x 11 mgHandbook1111* Provided in a 100 mg/ml Plasmid Purification Handbook 08/2003 QIAfilter Plasmid KitsQIAfilter Plasmid KitMidi (25)Midi (100)Maxi (10)Maxi (25)Mega (5)Giga (5)Catalog 10025100 QIAGEN -tip 500 1025 QIAGEN -tip 2500 5 QIAGEN -tip 10000 5 QIAfilter Midi25100 CartridgesQIAfilter Maxi 1025 CartridgesQIAfilter Mega- 55 Giga Cartridges*Caps for QIAfilter 251001025 Buffer P11 x 110 ml 4 x 110 ml 1 x 110 ml 1 x 280 ml 2 x 140 ml1 x 700 mlBuffer P21 x 110 ml 4 x 110 ml 1 x 110 ml 1 x 280 ml 2 x 140 ml1 x 700 mlBuffer P31 x 110 ml 4 x 110 ml 1 x 110 ml 1 x 280 ml 2 x 140 ml1 x 700 mlBuffer FWB2 2 x 140 ml2 x 140 mlBuffer QBT1 x 110 ml 4 x 110 ml 1 x 110 ml 1 x 280 ml 1 x 200 ml1 x 400 mlBuffer QC3 x 190 ml 12 x 190 ml 3 x 240 ml 2 x 850 ml 5 x 220 ml 3 x 1000 ml.

6 1 x 500 mlBuffer QF1 x 170 ml 4 x 170 ml 1 x 170 ml 1 x 420 ml 1 x 200 ml1 x 510 mlRNase A 1 x 11 mg 4 x 11 mg 1 x 11 mg 1 x 28 mg 2 x 14 mg1 x 70 mgHandbook111111*The QIAfilter Mega-Giga Cartridge is designed for use with a 1 liter, 45 mm-neck glass bottle ( Schott, cat. no. 21810154, or Corning, cat. no. 1395-1L). Note: Bottle is not included. Provided in a 100 mg/ml Plasmid Purification Handbook 08/20037 EndoFree Plasmid KitsEndoFree Plasmid KitMaxi (10)Mega (5)Giga (5)Catalog 50010 QIAGEN -tip 2500 5 QIAGEN -tip 10000 5 QIAfilter Maxi10 CartridgesQIAfilter Mega-Giga 55 Cartridges*Caps for QIAfilter 10 Buffer P11 x 110 ml2 x 140 ml1 x 700 mlBuffer P21 x 110 ml2 x 140 ml1 x 700 mlBuffer P31 x 110 ml2 x 140 ml1 x 700 mlBuffer FWB2 2 x 140 ml2 x 140 mlBuffer QBT1 x 110 ml1 x 200 ml1 x 400 mlBuffer QC3 x 240 ml5 x 220 ml3 x 1000 ml,1 x 500 mlBuffer QN1 x 170 ml1 x 200 ml1 x 510 mlBuffer ER1 x 30 ml1 x 80 ml1 x 200 mlBuffer TE1 x 30 ml1 x 110 ml1 x 110 mlEndotoxin-free water 1 x 17 ml1 x 17 ml1 x 17 mlfor 70% ethanolRNase A 1 x 11 mg2 x 14 mg1 x 70 mgHandbook111 Certificate of Analysis111*The QIAfilter Mega-Giga Cartridge is designed for use with a 1 liter, 45-mm-neck glass bottle ( Schott, cat.)

7 No. 21810154, or Corning, cat. no. 1395-1L). Note: Bottle is not included. Provided in a 100 mg/ml Plasmid Purification Handbook 08/2003 Storage QIAGEN -tips and QIAfilter Cartridges should be stored dry and at room temperature(15 25 C). They can be stored for at least 2 years without showing any reduction inperformance, capacity, or quality of , QIAfilter, and EndoFree Plasmid Kits should be stored at room temperature(15 25 C). After adding RNase A, Buffer P1 should be stored at 2 8 C and is stablefor 6 months. Other buffers and RNase A stock solution can be stored for 2 years atroom temperature (15 25 C).Technical AssistanceAt QIAGEN we pride ourselves on the quality and availability of our technical Technical Service Departments are staffed by experienced scientists with extensivepractical and theoretical expertise in molecular biology and the use of QIAGEN you have any questions or experience any difficulties regarding any aspect of QIAGEN ,QIAfilter, or EndoFree Plasmid Kits, or QIAGEN products in general, please do not hesitate to contact us.

8 QIAGEN customers are a major source of information regarding advanced or specializeduses of our products. This information is helpful to other scientists as well as to theresearchers at QIAGEN . We therefore encourage you to contact us if you have anysuggestions about product performance or new applications and technical assistance and more information please call one of the QIAGEN TechnicalService Departments or local distributors (see inside back cover).Product Warranty and Satisfaction GuaranteeQIAGEN guarantees the performance of all products in the manner described in ourproduct literature. The purchaser must determine the suitability of the product for its particular use. Should any product fail to perform satisfactorily due to any reason otherthan misuse, QIAGEN will replace it free of charge or refund the purchase price.

9 Wereserve the right to change, alter, or modify any product to enhance its performanceand design. If a QIAGEN product does not meet your expectations, simply call yourlocal Technical Service Department. We will credit your account or exchange the product as you wish. A copy of QIAGEN terms and conditions can be obtained on request, and is also provided on the back of our invoices. If you have questions about product specificationsor performance, please call QIAGEN Technical Service or your local distributor (seeinside back cover). QIAGEN Plasmid Purification Handbook 08/20039 Product Use LimitationsQIAGEN, QIAfilter, and EndoFree Plasmid Kits are developed, designed and sold forresearch purposes only. They are not to be used for human diagnostic or drug purposesor to be administered to humans unless expressly cleared for that purpose by the Foodand Drug Administration in the USA or the appropriate regulatory authorities in thecountry of use.

10 All due care and attention should be exercised in the handling of manyof the materials described in this InformationWhen 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 PDFformat at you can find, view, and print the MSDSfor each QIAGEN kit and kit component. The following risk and safety phrases apply to components of the QIAGEN Plasmid Kits,QIAfilter Plasmid Kits, and EndoFree Plasmid P2 Contains sodium hydroxide: irritant. Risk and safety phrases:* R36/38, S13-26-36-46 Buffer P3 Contains acetic acid: irritant. Risk and safety phrases:* R36/38, S13-26-36-46 Buffers QBT, QC, QF, and QNContain isopropanol: flammable.