QIAprep® Miniprep Handbook - University of San Diego Home Pages
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1. Very large plasmids and cosmids are often maintained at very low copy numbers per cell Table 5 Origins of replication and copy numbers of various plasmids 3 Origin of DNA construct replication Copy number Classification Plasmids pUC vectors pMB 1 500 700 High copy pBluescript vectors ColE1 300 500 High copy pGEM vectors pMB 1 300 400 High copy pTZ vectors pMB 1 gt 1000 High copy pBR322 and derivatives pMBI1 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 QlAprep Miniprep Handbook 05 2012 29 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 DH5a 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 endonuc2. centrifugation should be performed at 3000 x g for 1 min and 8 The flow through does not need to be discarded Step 9 Transfer the QlAprep spin column to a microcentrifuge tube Centrifuge at maximum speed for 1 min Continue with step 10 of the protocol 20 QlAprep Miniprep Handbook 05 2012 Protocol Plasmid DNA Purification using the QlAprep Spin Miniprep Kit and a Vacuum Manifold This protocol is designed for purification of up to 20 pg high copy plasmid DNA from 1 5 ml overnight cultures of E coli grown in LB medium using QlAprep spin columns on QlAvac 24 Plus or other vacuum manifolds with luer connectors For purification of low copy plasmids and cosmids large plasmids gt 10 kb and DNA prepared using other methods refer to the recommendations on page 34 Please read Important Notes on pages 12 18 before starting Note All protocol steps should be carried out at room temperature 15 25 C Procedure 1 Resuspend pelleted bacterial cells in 250 pl Buffer P1 and transfer to a micro centrifuge tube Ensure that RNase A has been added to Buffer P1 No cell clumps should be visi ble after resuspension of the pellet If LyseBlue reagent has been added to Buffer P1 vigorously shake the buffer bot tle 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 pl Buffer P2 and mix thoroughl
3. 8051 for use with the Matrix pipet mentioned above These can be purchased from the supplier listed above E Finntip Multistepper pipet tips for use with single channel pipets These are available from Thermo Electron Corporation www thermo com Guidelines for QlAvac manifolds QlAvac 24 Plus and QlAvac 96 facilitate DNA minipreps by providing a convenient modular vacuum manifold for use with the QlAprep system The following recommendations should be followed when handling QlAvac manifolds HM QlAvac 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 BE Always store QlAvac manifolds clean and dry To clean simply rinse all compo nents 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 BE Always place the QlAvac manifold on a secure bench top or work area If dropped the manifold may crack E The components of QlAvac manifolds are not resistant to ethanol methanol or other organic solvents Table 4 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 QlAprep Miniprep Handbook 05 2012 15 MH To ensure consistent performance do not apply silicone or vacuum grease to any part of a QlAvac manifold The
4. QlAprep purified DNA Plasmid DNA prepared using the QlAprep system is suitable for a variety of routine applications including E Restriction enzyme digestion E Sequencing E Library screening E Ligation and transformation MH In vitro translation E Transfection of robust cells Principle The QlAprep 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 QlAprep Miniprep Kits completely replaces glass or silica slurries for plasmid minipreps The procedure consists of three basic steps E Preparation and clearing of a bacterial lysate BE Adsorption of DNA onto the QlAprep membrane BE Washing and elution of plasmid DNA All steps are performed without the use of phenol chloroform CsCl ethidium bromide and without alcohol precipitation Preparation and clearing of bacterial lysate The QlAprep miniprep procedure uses the modified alkaline lysis method of Birnboim and Doly 2 Bacteria are lysed under alkaline conditions and the lysate is subse quently neutralized and adjusted to high salt binding conditions in one step After lysate clearing the sample is ready for purification on the QlAprep silica membrane For more details on growth of bacterial cultures and alkaline lysis please refer to Appendix A starting on page 29 In the QlAprep Spin procedure lysates are cleared by centrifugation while the QlAprep 96 T
5. 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 propagate b Lysate prepared incorrectly c Buffer P2 precipitated d Cell resuspension incomplete Read Growth of bacterial cultures page 29 and check that the conditions for optimal growth were met Check storage conditions and age of buffers Redissolve by warming to 37 C Pelleted cells should be completely resuspended in Buffer P1 Do not add Buffer P2 until an even suspen sion is obtained DNA is found in the flow through of cleared lysate a QlAprep membrane overloaded 26 If rich culture media such as TB or 2x YT are used culture volumes must 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 31 QlAprep Miniprep Handbook 05 2012 Comments and suggestions 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 insufficient containing RNase A is 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 wash buffer Buffer PE Little or no DNA in eluate a Elution buffer incorrect DNA is eluted only in the presence of l
6. 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 CO 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 pl 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 11 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 QlAprep 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 QlAprep modules indicated in Table 2 on the QlAvac manifold The vacuum pressure is the pressure differential between the inside of th
7. for all strains BE When plasmid or cosmids are gt 10 kb pre heat Buffer EB or water to 70 C prior to eluting DNA from the QlAprep membrane A 10 ml overnight LB culture typically yields 5 10 pg 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 gt 50 kb Plasmids that are gt 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 DNAfree 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 37 Purification of plasmid DNA prepared by other methods Plasmid DNA isolated by other methods can be further purified using QlAprep modules and any of the QlAprep protocols in this handbook C1 Add 5 volumes of Buffer PB to 1 volume of the DNA solution and mix e g add 500 pl Buffer PB to 100 pl of DNA sample C2 Apply the samples to QlAprep spin columns or to the wells of a QlAprep 96 well plate Draw the samples through the QlAprep membrane by centri
8. handling options to suit every throughput need Low throughput The QlAprep Spin Miniprep Kit is designed for quick and convenient processing of 1 24 samples simultaneously in less than 30 minutes QlAprep spin columns can be used in a microcentrifuge or on any vacuum manifold with luer connectors e g QlAvac 24 Plus The QlAprep Spin Miniprep Kit can be fully automated on the QlAcube The innovative QlAcube uses advanced technology to process QIAGEN spin columns enabling seamless integration of automated low throughput sample prep into your laboratory workflow Sample preparation using the QlAcube follows the same steps as the manual procedure i e lyse bind wash and elute enabling you to continue using the QlAprep Spin Miniprep Kit for purification of high quality plasmid DNA The QlAcube is 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 giagen com My lAcube QlAprep Miniprep Handbook 05 2012 7 High throughput The QlAprep 96 Turbo Miniprep Kit enables up to 96 minipreps to be performed simultaneously in less than 45 minutes on the QlAvac 96 For automated high through put plasmid purification the QlAprep 96 Turbo BioRobof Kit enables up to 96 minipreps to be processed in 70 minutes Applications using
9. if cells were not harvested in this block provided with the kit Ensure that RNase A has been added to Buffer P1 No cell clumps should be visible after resuspension of the pellet 2 Add 250 pl Buffer P2 to each sample Dry the top of the flat bottom 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 QlAvac 96 see pages 13 and 15 17 Place the TurboFilter 96 plate in the QlAvac top plate make sure that the plate is seated securely Seal unused wells of the TurboFilter with tape Place the plate holder inside the QlAvac base Place QlAprep 96 plate into the plate holder E Place QlAvac 96 top plate squarely over base The QlAprep plate should now be positioned under the TurboFilter plate Attach QlAvac to a vacuum source 3 Remove the tape from the block Add 350 pl Buffer N3 to each sample dry the top of the flat bottom 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 immedi ately after addition of Buffer N3 The solutions should become cloudy QlAprep Mini
10. of 50 pl and performing a short incubation after addition of the elution buffer 250 L 100 5 1 200 4 80 g F 5 150 4 F 60 5 100 4 L 202 z 9 50 F 20 T T T 50 100 150 Elution volume pl Figure 1 Elution volume versus DNA concentration and recovery Using the QlAprep Spin protocol 10 pg pUC18 DNA was purified and eluted with the indicated volumes of Buffer EB The standard protocol uses 50 pl Buffer EB for elution since this combines high yield with high concentration However the yield can be increased by increasing the elution volume 100 u E E z n a 80 ee ween m m 60 gt 100 pl g 50ypl amp 40 20 o T T if T T T 1 2 3 4 5 10 30 Incubation time min Figure 2 Incubation time versus DNA recovery Using the QlAprep Spin Miniprep protocol 10 pg pBluescript DNA was purified and eluted after the indicated incubation times with either 50 pl or 100 pl Buffer EB The graph shows that an incubation time of 1 minute and doubling the elution buffer volume increases yield EEE SSS SSS SSS 10 QlAprep Miniprep Handbook 05 2012 Table 1 Effect of different compositions of growth medium LB on DNA yield Culture media Yield LB containing 10 g liter NaCl 11 5 pg LB containing 5 g liter NaCl 9 5 pg QlAprep Spin Miniprep Kit was used to purify DNA from 1 5 ml LB overnight cultures of XL1 Blue containing pBluescript Elution was performed ac
11. spring lock on the top plate and the self sealing gasket QlAvac 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 Chemical resistance properties of QlAvac 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 QlAvac 24 Plus is resistant to these chemicals QlAvac vacuum manifolds QlAvac 24 Plus Manifold Figure 3 Components of the QlAvac 24 Plus manifold 1 QlAvac 24 Plus vacuum manifold 2 Luer slot closed with luer plug 3 Spin column 16 QlAprep Miniprep Handbook 05 2012 QlAvac 96 Manifold Figure 4 Components of the QlAvac 96 manifold 1 QlAvac base which holds a waste tray a plate 4 QlAvac 96 top plate with aperture for 96 well holder or a microtube rack plate 2 Waste tray 5 Microtube rack 3 Plate holder shown with 96 well plate 6 96 well plate Not included with QlAvac 96 Included in QlAprep 96 Turbo Miniprep Kits QlAprep Miniprep Handbook 05 2012 17 QlAprep Spin Procedure in microcentrifuges on vacuum manifolds Pelleted bacteria Resuspend Lyse Neutralize Bind Bind Li Vacuum Wash Wash Pure plasmid
12. 5 2012 Storage QlAprep 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 Pl is stable for 6 months when stored at 2 8 C RNase A stock solution can be stored for two years at room temperature Intended Use QlAprep Miniprep Kits are intended for molecular biology applications These products are not intended for the diagnosis prevention or treatment of a disease 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 material safety data sheets MSDSs These are available online in convenient and compact PDF format at www giagen 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
13. 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 mini mizing 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 fragmen
14. DNA Pure plasmid DNA 18 QlAprep Miniprep Handbook 05 2012 Protocol Plasmid DNA Purification using the QlAprep Spin Miniprep Kit and a Microcentrifuge This protocol is designed for purification of up to 20 pg 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 gt 10 kb and DNA prepared using other methods refer to the recommendations on page 34 Please read Important Notes on pages 12 18 before starting Note All protocol steps should be carried out at room temperature 15 25 C Procedure 1 Resuspend pelleted bacterial cells in 250 pl Buffer P1 and transfer to a micro centrifuge 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 pl Buffer P2 and mix thoroughly by inverting the tube 4 6 times Mix gently by inverting the tube Do not vortex as 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 suspens
15. Second Edition May 2012 QlAprep Miniprep Handbook For purification of molecular biology grade DNA Plasmid Large plasmids gt 10 kb Low copy plasmids and cosmids Plasmid DNA prepared by other methods QIAGEN QIAGEN Sample and Assay Technologies QIAGEN is the leading provider of innovative sample and assay technologies enabling the isolation and detection of contents of any biological sample Our advanced high quality products and services ensure success from sample to result QIAGEN sets standards in Purification of DNA RNA and proteins Nucleic acid and protein assays microRNA research and RNAi Automation of sample and assay technologies Our mission is to enable you to achieve outstanding success and breakthroughs For more information visit www giagen com Contents Kit Contents 4 Storage 5 Intended Use 5 Safety Information 5 Quality Control 6 Introduction 7 Principle 8 Using LyseBlue reagent 1 Important Notes 12 Guidelines for QlAvac manifolds 15 Protocols EM Plasmid DNA Purification using the QlAprep Spin Miniprep Kit and a microcentrifuge 19 and 5 ml collection tubes 20 and a vacuum manifold 21 Plasmid DNA Purification using the QlAprep 96 Turbo Miniprep Kit 23 Troubleshooting Guide 26 Appendix A Background Information 29 Growth of bacterial cultures 29 Preparation of cell lysates 32 Appendix B Agarose Gel Analysis of Plasmid DNA 33 Appendix C Special Applications 34 Purification of
16. ck on the base making sure that the QlAprep plate is positioned securely 11 To elute DNA add 100 pl of Buffer EB 10 mM Tris Cl pH 8 5 or water to the center of each well of the QlAprep plate let stand for 1 min and apply maximum vacuum for 5 min Switch off vacuum and ventilate QlAvac 96 slowly For increased DNA concentration an elution volume of 75 pl can be used 2 gt me me Se on gt QlAprep Miniprep Handbook 05 2012 25 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 giagen 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 protocols in this handbook or sample and assay technologies for contact information see back cover or visit www giagen com Low or no yield General Comments and suggestions 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 5 page 33 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
17. cording to the standard protocol 50 pl Buffer EB and 1 min incuba tion Use of the recommended LB composition with 10 g liter NaCl also see Appendix A p 43 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 P 1 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 pl 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 homo geneously 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 ho
18. croporous tape sheets for covering 19571 50 96 well 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 QlAprep Miniprep Handbook 05 2012 39 Notes 40 QlAprep Miniprep Handbook 05 2012 Notes QlAprep Miniprep Handbook 05 2012 41 Notes 42 QlAprep Miniprep Handbook 05 2012 Trademarks QIAGEN QlAcube QlAprep BioRobot DirectPrep LyseBlue R E A L TurboFilter QIAGEN Group DHS Life Technologies Inc Heraeus Heraeus Holding GmbH pBluescript Agilent Technologies Inc pGEM Promega Corp Beckman Beckman Instruments Inc Finntip Multistepper Thermo Electron Oy Corporation Limited License Agreement for QlAprep 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
19. e 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 37 Vacuum recommendations are given in negative units Table 2 to indicate the required reduction in pressure with respect to the atmosphere Table 3 provides pressure conversions to other units Use of a vacuum pressure lower than recommended may reduce DNA yield and purity QlAprep Miniprep Handbook 05 2012 13 Table 2 Regulation of vacuum pressures for QlAprep multiwell procedures Procedure Vacuum Module used for Vacuum pressure manifold checking pressure mbar mm Hg QlAprep 96 Turbo QlAvac 96 QlAprep 96 plate 40 to 200 30 to 150 Pressure should be regulated using empty modules on the manifold t Values apply to empty modules on QlAvac During the working procedure the vacuum may exceed the values indicated Table 3 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 E Ensure that the elution buffer is dispensed directly onto the center of the QlAprep membrane for optimal elution of DNA Average eluate volume is 48 pl from an elution buffer volume of 50 pl QlAprep spin procedures and 60 pl from an elution buf
20. ed low throughput plasmid purification QlAcube 110 V Robotic workstation for automated purification 9001292 QlAcube 230 V t of nucleic acids or proteins using QIAGEN 90012938 spin column kits 1 year warranty on parts and labort Accessories Starter Pack QlAcube Pack includes reagent bottle racks 3 rack 990395 labeling strips 8 200 pl filter tips 1024 1000 pl filter tips 1024 1000 pl filter ips wide bore 1024 30 ml reagent bottles 18 rotor adapters 240 rotor adapter holder Individual Buffers and accessories Buffer N3 500 ml Buffer N3 19064 Buffer PB 500 ml Buffer PB 19066 Buffer PE 100 ml Buffer PE concentrate 19065 concentrate RNase A 250 mg RNase A 70 U mg 100 mg ml 19101 Collection Tubes 1000 collection tubes 2 ml 19201 2 ml Collection Microtubes Nonsterile polypropylene tubes 1 2 ml 19560 racked 960 in racks of 96 US Canada and Japan t Rest of world Agreements for comprehensive service coverage are available please inquire 38 QlAprep Miniprep Handbook 05 2012 Ordering Information Product Contents Cat no Collection Microtube Nonsterile polypropylene caps for collection 19566 Caps collection microtubes 1 2 ml 960 in strips of 8 loose in bag Flat Bottom Blocks 24 96 well blocks with 2 ml wells 24 blocks 19572 per case Tape Pads 5 Adhesive tape sheets for sealing 19570 multiwell plates and blocks 25 sheets per pad 5 pads per pack AirPore Tape Sheets Mi
21. fer volume of 100 pl QlAprep multiwell procedures E For increased DNA yield use a higher elution buffer volume For increased DNA concentration use a lower elution buffer volume see DNA yield page 9 BE 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 lt 7 0 can decrease yield Note Store DNA at 20 C when eluted with water as DNA may degrade in the absence of a buffering agent BE 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 14 QlAprep Miniprep Handbook 05 2012 Multichannel pipet recommendations Many steps of the QlAprep 96 Turbo procedure 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 Pipet tip recommendations Some standard 1 ml pipet tips are not easily accommodated in the flat bottom blocks that are used in the QlAprep 96 Turbo Miniprep protocol When pipetting into flat bottom blocks we recommend using pipet tips with 1 25 ml or 1 5 ml fill volume such as E Matrix pipet tips cat no
22. fugation or vacuum and continue the appropriate protocol at the Buffer PE wash step The optional wash step with Buffer PB is not necessary 34 QlAprep Miniprep Handbook 05 2012 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 Ausubel F M et al eds 1991 Current protocols in molecular biology Wiley Interscience New York 5 Birnboim H C 1983 A rapid alkaline extraction method for the isolation of plasmid DNA Methods Enzymol 100 243 255 QlAprep Miniprep Handbook 05 2012 35 Ordering Information Product Contents Cat no QlAprep Spin For 50 plasmid minipreps 27104 Miniprep Kit 50 50 QlAprep Spin Columns Reagents Buffers Collection Tubes 2 ml QlAprep Spin For 250 plasmid minipreps 27106 Miniprep Kit 250 250 QlAprep Spin Columns Reagents Buffers Collection Tubes 2 ml QlAprep 96 Turbo For 4 x 96 plasmid minipreps LYN Miniprep Kit 4 A Turbofilter 96 Plates 4 QlAprep 96 Plates 4 Flat Bottom Blocks with Lids Reagents Buffers Collection Microtubes 1 2 ml Caps QlAprep 96 Turbo For 24 x 96 Plasmid minipreps 27193 Minip
23. gh levels of nuclease activity or high carbohydrate content Host strains such as XL 1 Blue and DH5 do not require this additional wash step a A a D a lt 8 Wash QlAprep 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 QlAprep column in a clean 1 5 ml microcentrifuge tube To elute DNA add 50 pl Buffer EB 10 mM Tris Cl pH 8 5 or water to the center of each QlAprep spin column let stand for 1 min and centrifuge for 1 min Protocol Plasmid DNA Purification using the QlAprep Spin Miniprep Kit and 5 ml Collection Tubes The QlAprep 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 19 20 should be followed with the following modifications Step 4 Place a QlAprep 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 For washing steps
24. ice ca giagen com China techservice cn giagen com Denmark techservice nordic qiagen com Finland techservice nordic qiagen com France techservice fr qiagen com Germany techservice de qiagen com Hong Kong techservice hk qiagen com India techservice india giagen com Ireland techservice uk giagen com Italy techservice it qiagen com Japan techservice jp giagen com Korea South techservice kr giagen com Luxembourg techservice bnl giagen com Mexico techservice mx qiagen com The Netherlands techservice bnl giagen com Norway techservice nordic qiagen com Singapore techservice sg qiagen com Sweden techservice nordic qiagen com Switzerland techservice ch giagen com UK techservice uk giagen com USA techservice us giagen com 1071266 05 2012 QIAGEN Sample amp Assay Technologies
25. icrotubes RS Caps DirectPrep 96 For 4 x 96 plasmid Minipreps 962341 BioRobot Kit A A DirectPrep 96 Plates Reagents Buffers Flat Bottom Blocks and Lids 96 Well Microplates RB AirPore Tape Sheets Tape Pads Related products for BAC PAC P1 purification QIAGEN Large 10 QIAGEN ip 500 Reagents Buffers 12462 Construct Kit 10 ATP Dependent Exonuclease QlAvac and accessories QlAvac 24 Plus Vacuum manifold for processing 1 24 spin 19413 columns includes QlAvac 24 Plus Vacuum Manifold Luer Plugs Quick Couplings QlAvac 96 Vacuum manifold for processing QIAGEN 19504 96 well plates includes QlAvac 96 Top Plate Base Waste Tray Plate Holder Rock of Collection Microtubes 1 2 ml QlAvac Luer For processing 1 24 QlAprep Spin Columns 19541 Adapter Set 6 adapters each with 4 luer connectors 24 plugs Requires use of QlAvac Multiwell Larger kit sizes available please inquire t For use with BioRobot 3000 or 8000 workstations Larger kit sizes available please inquire ATP solution required for exonuclease digestion is not provided Compatible only with QlAvac Top Plates containing flip up lid QlAprep Miniprep Handbook 05 2012 37 Ordering Information Product Contents Cat no Vacuum Regulator For use with QlAvac manifolds 19530 Vacuum Pump Universal vacuum pump 84000 100 V 50 60 Hz Vacuum Pump Universal vacuum pump 84010 115 V 60 Hz Vacuum Pump Universal vacuum pump 84020 230 V 50 Hz Automat
26. ion will turn blue after addi tion 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 pl Buffer N3 and 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 25 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 sus pension 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 EEE QlAprep Miniprep Handbook 05 2012 19 2 gt me me n EL 5 5 Apply the supernatants from step 4 to the QlAprep spin column by decanting or pipetting 6 Centrifuge for 30 60 s Discard the flow through 7 Recommended Wash the QlAprep 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 wildtype strain which have hi
27. lease 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 DH5a 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 since 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 which ensures that the plasmids segregate equally during cell division Daughter cells that do not receive plasmids will replicate much faste
28. low copy plasmids and cosmids 34 Purification of plasmid DNA prepared by other methods 34 References 35 Ordering Information 36 QlAprep Miniprep Handbook 05 2012 3 Kit Contents QlAprep Spin Miniprep Kit 50 250 Catalog no 27104 27106 QlAprep Spin Columns 50 250 Buffer P1 20 ml 1x 20 ml 1 x 50 ml Buffer P2 20 ml 1x 20 ml 1 x 50 ml Buffer N3 30 ml 140 ml Buffer PB 30 ml 150 ml Buffer PE concentrate 2x6nml 55 ml Buffer EB 15 ml 55 ml LyseBlue 20 pl 1 x 20 pl 1 x 50 pl RNase At 2 mg 1x2 mg 1x5 mg Collection Tubes 2 ml 50 250 Quick Start Protocol 1 1 QlAprep 96 Turbo Miniprep Kit 4 24 Catalog no 27191 27193 TurboFilter 96 Plates 4 24 QlAprep 96 Plates 4 24 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 2x55 ml 1 x 55 ml 2 x 250 ml RNase At 1x 125 pl 1x 15 mg 3 x 25 mg Tape Pads 1 6 Rack of Collection Microtubes 1 2 ml 4 24 Caps for Collection Microtubes 55x8 6x55x8 Flat Bottom Blocks and Lids 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 5 for safety information t Provided as a 100 mg ml solution 4 QlAprep Miniprep Handbook 0
29. mogeneously 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 QlAprep Miniprep Handbook 05 2012 11 Important Notes Please read the following notes before starting any of the QlAprep 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 plas mid 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 gt 8000 rpm 6800 x g in a con ventional 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 QlAprep Turbo 96 1 Fill each well of a 96 well flat bottom block with 1 3 ml of growth medium contain ing the appropriate selective agent Inoculate each well from a single bacterial colony Incubate the cultu
30. ng vacuum Repeat once e Me r a a 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 QlAvac 96 slowly Lift the top plate from the base not the QlAprep 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 QlAprep 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 QlAprep plate Residual ethanol from Buffer PE may inhibit subsequent enzymatic reactions 24 QlAprep Miniprep Handbook 05 2012 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 QlAprep 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 QlAvac 96 Place a 96 well microplate directly on the rack Place the top plate ba
31. ning RNase A is more 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 incorrectly Buffer P2 to prevent shearing Reduce culture volume if lysate is too viscous for gentle mixing b Buffer N3 added Upon addition of Buffer N3 in step 3 mix immediately incorrectly but gently c Lysis too long Lysis in step 2 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 28 QlAprep Miniprep Handbook 05 2012 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 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 which 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
32. olumns QlAvac 24 Plus see pages 13 and 15 17 E Ensure that the vacuum source is connected to the upper threaded hole of the QlAvac 24 Plus and the lower threaded hole is tightly sealed using the screw cap E If using the QlAvac Connecting System connect the system to the manifold and vacuum soured as described in the QlAvac 24 Plus Handbook E Insert up to 24 spin columns into the luer slots of the QlAvac 24 Plus Close unused luer slots with luer plugs Other vacuum manifolds Follow the supplier s instructions Insert each QlAprep column into a luer connector A AO 9 0 55 lt 3 to go 5 Apply the supernatant from step 4 to the QlAprep spin column by decanting or pipetting 6 Switch on vacuum source to draw the solution through the QlAprep spin columns and then switch off vacuum source 7 Recommended Wash the QlAprep spin column by adding 0 5 ml Buffer PB Switch on vacuum source After the solution has moved through the column switch off vacuum source 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 DH5a do not require this additional wash step 8 Wash the QlAprep 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 vacu
33. on 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 QlAprep membrane If excess culture volume is used alkaline lysis will be inefficient the QlAprep 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 7 Recommended composition of Luria Bertani medium Contents Per liter Tryptone 10g Yeast extract 5g NaCl 10g QlAprep Miniprep Handbook 05 2012 31 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
34. ow 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 QlAprep dispensed onto membrane to ensure that the buffer completely covers membrane the surface of the membrane for maximum elution efficiency Low DNA quality DNA does not perform well in downstream applications a Eluate salt concentration For the QlAprep spin column modify the wash step by too high incubating the column for 5 minutes at room temperature 15 25 C after adding 0 75 ml of Buffer PE and then centrifuging For QlAprep 96 Turbo preparations ensure that two 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 residual Ensure that step 9 in the QlAprep Spin Miniprep protocol ethanol and steps 9 and 10 in the QlAprep 96 Turbo Miniprep protocol are performed QlAprep Miniprep Handbook 05 2012 27 Comments and suggestions 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 insufficient contai
35. prep Handbook 05 2012 23 2 gt me me Se gt fe 4 Remove the tape from the block Pipet the lysates from step 3 850 pl 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 37 between the QlAvac and the vacuum source 5 Switch off vacuum and ventilate the QlAvac 96 slowly Discard the Turbofilter plate Transfer the QlAprep plate containing the cleared lysates to the top plate of the manifold Seal any unused wells of the QlAprep plate with tape Replace plate holder in the base with waste tray Place the top plate squarely over the base making sure that the QlAprep plate is seated securely Apply vacuum The flow through is collected in the waste tray 6 Recommended Switch off vacuum and wash QlAprep plate by adding 0 9 ml Buffer PB to each well and applying vacuum This step is necessary to remove trace nuclease activity when using endA strains such as the JM series HB101 and its derivatives or any wildtype strain which have high levels of nuclease activity or high carbohydrate content Host strains such as XL 1 Blue and DH5a do not require this additional step 7 Switch off vacuum Wash QlAprep plate by adding 0 9 ml of Buffer PE to each well and applyi
36. r 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 B 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 6 are based on these considerations 30 QlAprep Miniprep Handbook 05 2012 Table 6 Concentrations of commonly used antibiotics Stock solutions Working concentration Antibiotic Concentration Storage dilution Ampicillin 50 mg ml in water 20 C 100 pg ml 1 500 sodium salt Chloramphenicol 34 mg ml in ethanol 20 C 170 pg ml 1 200 Kanamycin 10 mg ml in water 20 C 50 pg ml 1 200 Streptomycin 10 mg ml in water 20 C 50 pg ml 1 200 Tetracycline HCl 5 mg ml in ethanol 20 C 50 pg ml 1 100 Culture media LB broth is the recommended culture medium for use with QlAprep Kits since richer broths such as TB Terrific Broth or 2x YT lead to extremely high cell densities which can overload the purificati
37. rep Kit 24 24 x TurboFilter 96 Plates 24 x QlAprep 96 Plates 24 FlatBottom Blocks with Lids Reagents Buffers Collection Microtubes 1 2 ml Caps QlAprep 96 Turbo For 4 x 96 plasmid minipreps 962141 BioRobot Kit 4 4 each TurboFilter 96 and QlAprep 96 Plates Flat Bottom Blocks and Lids Reagents Buffers Collection Microtubes 1 2 ml Caps 96 Well Microplates RB and Lids Tape Pads QIAGEN Plasmid Plus 96 Miniprep and BioRobot Kits for purification of transfection grade plasmid DNA in 96 well format QIAGEN For 4 x 96 plasmid minipreps TurboFilter 96 960241 Plasmid Plus 96 Plates and Plasmid Plus 96 Plates Buffers BioRobot Kit 4 Reagents Flat Bottom Blocks S Blocks and Elution Microtubes for use with the BioRobot Universal System QIAGEN For 4 x 96 plasmid minipreps TurboFilter 96 16181 Plasmid Plus 96 Plates Plasmid Plus 96 Plates Buffers Reagents Miniprep Kit 4 Flat Bottom Blocks S Blocks and Elution Microtubes requires use of QlAvac 96 and Elution Microtube Adapter or a centrifugation system suitable for 96 well blocks Requires the use of QlAvac 96 36 QlAprep Miniprep Handbook 05 2012 Ordering Information Product Contents Cat no DirectPrep 96 Kits for high throughput plasmid DNA purification DirectPrep 96 For 4 x 96 plasmid Minipreps 27361 Miniprep Kit 4 4 DirectPrep 96 Plates Reagents Buffers Flat Bottom Blocks and Lids Air Pore Tape Sheets Tape Pads Elution M
38. res for 20 24 h at 37 C with vigorous shaking The wells in the block may be protected against spillover by covering the block with a plastic lid or adhesive tape AirPore microporous tape sheets promote gas exchange during culturing see ordering information page 37 If non porous tape is used pierce 2 3 holes in the tape with a needle 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 remain ing droplets of medium WARNING Ensure that the buckets on the rotor have sufficient clearance to accom modate the 2 ml flatbottom blocks before starting the centrifuge le SSS SSS SSS SSS SSS SSS 12 QlAprep Miniprep Handbook 05 2012 Buffer notes Add the provided RNase A solution to Buffer Pl before use Use 1 vial RNase A centrifuge briefly before use per bottle Buffer P1 for a final concentration of 100 pg 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
39. tep 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 QlAprep column with 50 100 pl 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 20 C when eluted with water since DNA may degrade in the absence of a buffering agent DNA yield Plasmid yield with the QlAprep miniprep system varies depending on plasmid copy number per cell see page 29 the individual insert in a plasmid factors that affect growth of the bacterial culture see page 29 the elution volume Figure 1 and the elution incubation time Figure 2 A 1 5 ml overnight culture can yield from 5 to LyseBlue reagent is only supplied with the QlAprep Spin Miniprep Kit since multiwell or automated formats do not allow visual control of individual samples QlAprep Miniprep Handbook 05 2012 9 15 pg of plasmid DNA Table 1 page 11 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 31 eluting plasmid DNA in a volume
40. 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 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 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 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 giagen com 2002 2012 QIAGEN all rights reserved www qiagen com Australia techservice au giagen com Austria techservice at giagen com Belgium techservice bnl giagen com Brazil suportetecnico brasil qiagen com Canada techserv
41. ts in the supernatant Since chromosomal fragments are chemically indistinguishable from plasmid DNA under the conditions used the two species will not be separated on QlAprep 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 32 QlAprep Miniprep Handbook 05 2012 Appendix B Agarose Gel Analysis of Plasmid DNA The QlAprep Miniprep procedure can be analyzed using agarose gel electrophoresis as shown in Figure 5 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 5 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 5 Agarose gel analysis of the QlAprep Miniprep procedure C cleared lysate F flow through E eluted plasmid M markers QlAprep Miniprep Handbook 05 2012 33 Appendix C Special Applications Purification of low copy plasmids and cosmids All QlAprep 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 slight modifications to the protocols are required E The wash step with Buffer PB is required
42. um source 9 Transfer the QlAprep 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 QlAprep column in a clean 1 5 ml microcentrifuge tube To elute DNA add 50 pl Buffer EB 10 mM Tris Cl pH 8 5 or water to the center of the QlAprep spin column let stand for 1 min and centrifuge for 1 min 22 QlAprep Miniprep Handbook 05 2012 Protocol Plasmid DNA Purification using the QlAprep 96 Turbo Miniprep Kit This protocol is designed for high throughput plasmid DNA minipreps using TurboFilter 96 and QlAprep 96 plates on QlAvac 96 The kit accommodates up to 96 parallel preparations of up to 20 pg 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 a flatbottom block see page 12 for protocol For purification of low copy plasmids and cosmids large plasmids gt 10 kb and DNA prepared using other methods refer to the recommendations on page 34 DNA purification can be automated please call QIAGEN for more details Please read Important Notes on pages 12 18 before starting Note All protocol steps should be carried out at room temperature 15 25 C Procedure 1 Resuspend pelleted bacterial cells in 250 pl Buffer P1 and transfer to the flat bottom block
43. urbo Miniprep Kit provides TurboFilter strips or plates for lysate clearing by filtration 8 QlAprep Miniprep Handbook 05 2012 LyseBlue reagent Use of LyseBlue is optional and is not required to successfully perform plasmid preparations See Using LyseBlue reagent on page 11 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 QlAprep membrane QlAprep 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 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 s
44. 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 24 hour emergency information Emergency medical information in English French and German can be obtained 24 hours a day from Poison Information Center Mainz Germany Tel 49 6131 19240 QlAprep Miniprep Handbook 05 2012 5 Quality Control In accordance with QIAGEN s ISO certified Total Quality Management System each lot of the QlAprep Miniprep Kit is tested against predetermined specifications to ensure consistent product quality QlAprep Miniprep Handbook 05 2012 Introduction The QlAprep Miniprep system provides a fast simple and cost effective plasmid miniprep method for routine molecular biology laboratory applications QlAprep Miniprep Kits use silica membrane technology to eliminate the cumbersome steps associated with loose resins or slurries Plasmid DNA purified with QlAprep 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 QlAprep system consists of 2 products with different
45. y by inverting the tube gently 4 6 times Do not vortex as 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 addi tion 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 pl Buffer N3 and mix immediately and thoroughly by inverting the tube 4 6 times To avoid localized precipitation immediately after addition of Buffer N3 mix the solution gently but thoroughly Large culture volumes e g 25 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 sus pension 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 QlAprep Miniprep Handbook 05 2012 21 lt fe le c D Q 8 o 2 gt ne 7 2 5 During centrifugation prepare the vacuum manifold and QlAprep spin c
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