Qiagen Plasmid Purification Handbook

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Text of Qiagen Plasmid Purification Handbook

Second Edition

August 2003

QIAGEN Plasmid Purification HandbookQIAGEN Plasmid Midi, Maxi, Mega, and Giga Kits QIAfilter Plasmid Midi, Maxi, Mega, and Giga Kits EndoFree Plasmid Maxi, Mega, and Giga Kits For purification of ultrapure plasmid DNA

W W W. Q I A G E N . C O M

Trademarks and disclaimers Patented 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, Inc. DH5 is a trademark of Life Technologies, Inc. pBluescript is a registered trademark of Stratagene Inc. FPLC is a registered trademark of Amersham Biosciences. Triton is a registered trademark of Rohm and Haas Company. pGEM is a registered trademark of Promega Corp. Sorvall is a registered trademark of Sorvall Products, L.P. Purchase of QIAGEN products for PCR is accompanied by a limited license to use them in the Polymerase Chain Reaction (PCR) process for research and development activities in conjunction with a thermal cycler whose use in the automated performance of the PCR process is covered by the up-front license fee, either by payment to Applied Biosystems or as purchased, i.e. an authorized thermal cycler. The PCR process is covered by U.S. Patents 4,683,195 and 4,683,202 and foreign equivalents owned by Hoffmann-La Roche AG. Registered names, trademarks, etc. used in this document, even when not specifically marked as such, are not to be considered unprotected by law. 20002003 QIAGEN, all rights reserved.

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QIAGEN Plasmid Purification Handbook 08/2003

ContentsKit Contents Storage Technical Assistance Product Warranty and Satisfaction Guarantee Product Use Limitations Safety Information Introduction Comparison of QIAGEN large-scale plasmid purification options Principle and procedure Brief considerations for plasmid/cosmid purification procedures Protocols Plasmid or Cosmid DNA Purification Using QIAGEN Plasmid Midi and Maxi Kits Plasmid or Cosmid DNA Purification Using QIAGEN Plasmid Mega and Giga Kits Plasmid or Cosmid DNA Purification Using QIAfilter Plasmid Midi and Maxi Kits Plasmid or Cosmid DNA Purification Using QIAfilter Plasmid Mega and Giga Kits Plasmid or Cosmid DNA Purification Using the EndoFree Plasmid Maxi Kit Plasmid or Cosmid DNA Purification Using EndoFree Plasmid Mega and Giga Kits Very Low-Copy Plasmid/Cosmid Purification Using QIAGEN-tip 100 or QIAGEN-tip 500 Troubleshooting Guide Appendix A: Agarose Gel Analysis of the Purification Procedure Appendix B: Reliability of DNA Quantification by Spectrophotometry Appendix C: General Considerations for Optimal Results Growth of bacterial cultures Key steps in the plasmid purification protocols Removal of bacterial endotoxins Appendix D: Composition of Buffers Appendix E: QIAGEN Anion-Exchange Resin Appendix F: Purification of Plasmid DNA Prepared by Other Methods 16 21 26 31 36 41 47 52 60 62 63 63 69 71 74 76 79 5 8 8 8 9 9 10 10 13 13

QIAGEN Plasmid Purification Handbook 08/2003

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Appendix G: Removal of Endotoxins from Purified DNA Appendix H: Purification of M13 Replicative Form References Ordering Information QIAGEN Distributors

79 79 80 81 87

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QIAGEN Plasmid Purification Handbook 08/2003

Kit ContentsQIAGEN Plasmid KitsQIAGEN Plasmid Kit Catalog no. QIAGEN-tip 100 QIAGEN-tip 500 Buffer P1 Buffer P2 Buffer P3 Buffer QBT Buffer QC Buffer QF RNase A* Handbook Midi (25) 12143 25 1 x 110 ml 1 x 110 ml 1 x 110 ml 1 x 110 ml 3 x 190 ml 1 x 170 ml 1 x 11 mg 1 Midi (100) 12145 100 1 x 440 ml 1 x 440 ml 1 x 440 ml 1 x 440 ml 3 x 750 ml 2 x 280 ml 1 x 44 mg 1 Maxi (10) 12162 10 1 x 110 ml 1 x 110 ml 1 x 110 ml 1 x 110 ml 3 x 240 ml 1 x 170 ml 1 x 11 mg 1 Maxi (25) 12163 25 1 x 280 ml 1 x 280 ml 1 x 280 ml 1 x 280 ml 4 x 425 ml 1 x 420 ml 1 x 28 mg 1 Maxi (100) 12165 4 x 25 4 x 280 ml 4 x 280 ml 4 x 280 ml 4 x 280 ml 16 x 425 ml 4 x 420 ml 4 x 28 mg 1

QIAGEN Plasmid Kit Catalog no. QIAGEN-tip 2500 QIAGEN-tip10000 Buffer P1 Buffer P2 Buffer P3 Buffer QBT Buffer QC Buffer QF RNase A* Handbook

Mega (5) 12181 5 2 x 140 ml 2 x 140 ml 2 x 140 ml 1 x 200 ml 5 x 220 ml 1 x 200 ml 2 x 14 mg 1

Mega (25) 12183 25 2 x 700 ml 2 x 700 ml 2 x 700 ml 2 x 500 ml 5 x 1000 ml, 1 x 500 ml 2 x 510 ml 2 x 70 mg 1

Giga (5) 12191 5 1 x 700 ml 1 x 700 ml 1 x 700 ml 1 x 400 ml 3 x 1000 ml, 1 x 500 ml 1 x 510 ml 1 x 70 mg 1

Plasmid Buffer Set 19046 1 x 110 ml 1 x 110 ml 1 x 110 ml 1 x 110 ml 6 x 120 ml 2 x 85 ml 1 x 11 mg 1

* Provided in a 100 mg/ml solution.

QIAGEN Plasmid Purification Handbook 08/2003

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QIAfilter Plasmid KitsQIAfilter Plasmid Kit Catalog no. QIAGEN-tip 100 QIAGEN-tip 500 QIAGEN-tip 2500 QIAGEN-tip 10000 QIAfilter Midi Cartridges QIAfilter Maxi Cartridges QIAfilter MegaGiga Cartridges* Caps for QIAfilter Buffer P1 Buffer P2 Buffer P3 Buffer FWB2 Buffer QBT Buffer QC Buffer QF RNase A

Midi (25) 12243 25 25 25

Midi (100) 12245 100 100 100

Maxi (10) 12262 10 10 10

Maxi (25) 12263 25 25 25

Mega (5) 12281 5 5

Giga (5) 12291 5 5 1 x 700 ml 1 x 700 ml 1 x 700 ml 2 x 140 ml 1 x 400 ml

1 x 110 ml 4 x 110 ml 1 x 110 ml 1 x 280 ml 2 x 140 ml 1 x 110 ml 4 x 110 ml 1 x 110 ml 1 x 280 ml 2 x 140 ml 1 x 110 ml 4 x 110 ml 1 x 110 ml 1 x 280 ml 2 x 140 ml 2 x 140 ml

1 x 110 ml 4 x 110 ml 1 x 110 ml 1 x 280 ml 1 x 200 ml

3 x 190 ml 12 x 190 ml 3 x 240 ml 2 x 850 ml 5 x 220 ml 3 x 1000 ml, 1 x 500 ml 1 x 170 ml 4 x 170 ml 1 x 170 ml 1 x 420 ml 1 x 200 ml 1 x 11 mg 1 4 x 11 mg 1 x 11 mg 1 x 28 mg 2 x 14 mg 1 1 1 1 1 x 510 ml 1 x 70 mg 1

Handbook

* The QIAfilter Mega-Giga Cartridge is designed for use with a 1 liter, 45 mm-neck glass bottle (e.g. Schott, cat. no. 21810154, or Corning, cat. no. 1395-1L). Note: Bottle is not included.

Provided in a 100 mg/ml solution.

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QIAGEN Plasmid Purification Handbook 08/2003

EndoFree Plasmid KitsEndoFree Plasmid Kit Catalog no. QIAGEN-tip 500 QIAGEN-tip 2500 QIAGEN-tip 10000 QIAfilter Maxi Cartridges QIAfilter Mega-Giga Cartridges* Caps for QIAfilter Buffer P1 Buffer P2 Buffer P3 Buffer FWB2 Buffer QBT Buffer QC Buffer QN Buffer ER Buffer TE Endotoxin-free water for 70% ethanol RNase A Handbook Certificate of Analysis Maxi (10) 12362 10 10 10 1 x 110 ml 1 x 110 ml 1 x 110 ml 1 x 110 ml 3 x 240 ml 1 x 170 ml 1 x 30 ml 1 x 30 ml 1 x 17 ml 1 x 11 mg 1 1 Mega (5) 12381 5 5 2 x 140 ml 2 x 140 ml 2 x 140 ml 2 x 140 ml 1 x 200 ml 5 x 220 ml 1 x 200 ml 1 x 80 ml 1 x 110 ml 1 x 17 ml 2 x 14 mg 1 1 Giga (5) 12391 5 5 1 x 700 ml 1 x 700 ml 1 x 700 ml 2 x 140 ml 1 x 400 ml 3 x 1000 ml, 1 x 500 ml 1 x 510 ml 1 x 200 ml 1 x 110 ml 1 x 17 ml 1 x 70 mg 1 1

* The QIAfilter Mega-Giga Cartridge is designed for use with a 1 liter, 45-mm-neck glass bottle (e.g. Schott, cat. no. 21810154, or Corning, cat. no. 1395-1L). Note: Bottle is not included.

Provided in a 100 mg/ml solution.

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StorageQIAGEN-tips and QIAfilter Cartridges should be stored dry and at room temperature (1525C). They can be stored for at least 2 years without showing any reduction in performance, capacity, or quality of separation. QIAGEN, QIAfilter, and EndoFree Plasmid Kits should be stored at room temperature (1525C). After adding RNase A, Buffer P1 should be stored at 28C and is stable for 6 months. Other buffers and RNase A stock solution can be stored for 2 years at room temperature (1525C).

Technical AssistanceAt 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 any aspect of QIAGEN, QIAfilter, or EndoFree Plasmid Kits, or QIAGEN products in general, please do not hesitate to contact us. QIAGEN customers are a major source of information regarding advanced or specialized uses of our products. This information is helpful to other scientists as well as to the researchers at QIAGEN. We therefore encourage you to contact us if you have any suggestions about product performance or new applications and techniques. For technical assistance and more information please call one of the QIAGEN Technical Service Departments or local distributors (see inside back cover).

Product Warranty and Satisfaction GuaranteeQIAGEN guarantees the performance of all products in the manner described in our product 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 other than misuse, QIAGEN will replace it free of charge or refund the purchase price. We reserve the right to change, alter, or modify any product to enhance its performance and design. If a QIAGEN product does not meet your expectations, simply call your local 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 specifications or performance, please call QIAGEN Technical Service or your local distributor (see inside back cover).

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QIAGEN Plasmid Purification Handbook 08/2003

Product Use LimitationsQIAGEN, QIAfilter, and EndoFree Plasmid Kits are developed, designed and sold for research purposes only. They are not to be used for human diagnostic or drug purposes or to be administered to humans unless expressly cleared for that purpose by the Food and Drug Adm

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