CompactPrep Plasmid Purification Handbook
Contents
1. 10 CompactPrep Plasmid Purification Handbook 04 2012 Table 2 Composition of Luria Bertani medium Contents Per liter Tryptone 109 Yeast extract 5g NaCl 109 Preparation of LB medium Dissolve 10 g tryptone 5 g yeast extract and 10 g NaCl in 800 ml distilled water Adjust the pH to 7 0 with 1 N NaOH Adjust the volume to 1 liter with distilled water Sterilize by autoclaving Culture volume Do not exceed the maximum recommended culture volumes given at the beginning of each protocol Using larger culture volumes will lead to an increase in biomass and can affect the efficiency of alkaline lysis leading to reduced yield and purity of the preparation The protocol for QIAGEN plasmid kits is optimized for use with cultures grown in Luria Bertani LB medium grown to a cell density of approximately 3 4 x 10 cells ml We advise harvesting cultures after approximately 12 16 hours of growth which typically is the transition from logarithmic into stationary growth phase It is best to assess the cell density of the culture and if that is too high to reduce the culture volumes accordingly A high ratio of biomass to lysis buffers will result in poor lysis conditions and subsequently low DNA yield and purity For determination of cell density calibration of each individual spectrophotometer is required to facilitate accurate conversion of measurements into the number of cells per milliliter This can be ac2. Comments and suggestions Low or no yield No DNA in the cleared lysate before loading a Plasmid did not propagate Check that the conditions for optimal growth were met For more details see www qiagen com goto plasmidinfo b Alkaline lysis was If cells have grown to very high densities or a inefficient larger amount of culture medium than recommended was used the ratio of the biomass to lysis reagent is shifted This may result in poor lysis conditions because the volumes of Buffers P1 P2 and S3 are not sufficient for setting the plasmid DNA free efficiently Reduce the culture volume to improve the ratio of biomass to lysis buffer Also insufficient mixing of lysis reagents will result in reduced yield Mix thoroughly after addition of Buffers P1 P2 and P3 to achieve homogeneous suspensions Use LyseBlue to visualize efficiency of mixing c Insufficient lysis for low For low copy plasmid preparations doubling copy plasmids the volumes of Buffers P1 P2 S3 and BB may help to increase plasmid yield and quality d Buffer P2 or Buffer BB Redissolve by warming to 37 C precipitated e Cell Resuspension Pelleted cells should be completely incomplete resuspended in Buffer P1 Do not add Buffer P2 until an even suspension is obtained 20 CompactPrep Plasmid Purification Handbook 04 2012 Comments and suggestions DNA is found in the wash flow through Ethanol omitted from Repeat procedure with correctly prepared w
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4. trace of blue has gone and the suspension is colorless A homogeneous colorless suspension indicates that the SDS has been effectively precipitated 7 Centrifuge at 220 000 x g for 30 min at 4 C Remove supernatant containing plasmid DNA promptly Before loading the centrifuge the sample should be mixed again Centrifugation should be performed in non glass tubes e g polypropylene After centrifugation the supernatant should be clear 8 Centrifuge the supernatant again at 220 000 x g for 15 min at 4 C Remove supernatant containing plasmid DNA promptly This second centrifugation step should be carried out to avoid applying suspended or particulate material to the CompactPrep column Suspended material causing the sample to appear turbid can clog the CompactPrep column Optional Remove 35 or A 60 sample of the cleared lysate and save for an analytical gel to determine whether growth and lysis conditions were optimal 9 During incubation prepare the vacuum manifold and CompactPrep Midi or Maxi columns see vacuum manifolds pages 17 18 Potassium dodecyl sulfate CompactPrep Plasmid Purification Handbook 04 2012 17 10 Add 2 ml or A 5 ml Buffer BB to the lysate Mix by inverting 4 6 times and transfer the adjusted lysate into a tube extender attached to the CompactPrep column Buffer BB may precipitate when added to the cleared lysate after centrifugation at 4 Allow the lysate Buffer BB so
5. 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 QIAGEN specifically disclaims any other licenses expressed or implied other than those expressly stated QV GR 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 2012 QIAGEN all rights reserved A www qiagen com Australia techservice au qiagen com Austria techservice at qiagen com Belgium techservice bnI qiagen com Brazil suportetecnico brasil qiagen com Canada techservice ca qiagen com China techservice cn 2qiagen 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
6. April 2012 CompactPrep Plasmid Purification Handbook For preparation of molecular biology grade plasmid DNA from E coli QIAGEN Sample amp Assay Technologies 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 4 Intended Use 4 Safety Information 5 Quality Control 5 Introduction 6 Principle and procedure 6 Equipment and Reagents to Be Supplied by User 8 Important Notes 9 Protocol a Purification of Plasmid DNA using CompactPrep Plasmid Kits 15 Troubleshooting Guide 20 Appendix Agarose Gel Analysis of the Purification Procedure 22 Ordering Information 24 _ CompactPrep Plasmid Purification Handbook 04 2012 3 Kit Contents CompactPrep Plasmid Kit Midi 25 Maxi 25 Catalog no 12843 12863 CompactPrep Columns 25 Midi Columns 25 Maxi Columns Tube Extenders 10 ml 25 T
7. ash buffer wash buffer Buffer PE Low DNA quality Eluate contains residual Ensure that the CompactPrep column is dried ethanol sufficiently see step 13 page 22 or step 12 page 27 of the protocol CompactPrep column clogs during binding The amount of DNA in the Maximum binding capacity of the column has adjusted lysate exceeds been reached Remove residual lysate and the binding capacity of the perform all subsequent steps in a column microcentrifuge CompactPrep Plasmid Purification Handbook 04 2012 21 Appendix Agarose Gel Analysis of the Purification Procedure DNA yields and quality can be readily analyzed by agarose gel electrophoresis Poor yields and quality can be caused by a number of different factors To determine the stage of the procedure where the problem occurred save a fraction of the cleared lysate and analyze by agarose gel electrophoresis Preparation of samples Remove an aliquot from the cleared lysate as indicated in the protocol Precipitate the nucleic acids by adding 1 volume of isopropanol centrifuge for 15 min at maximum speed and discard supernatant Rinse the plasmid DNA pellets with 70 ethanol drain well and resuspend in 10 ul TE pH 8 0 L E MI 12 3 45 M2 Figure 2 Agarose gel analysis of the plasmid purification procedure Agarose gel analysis Run 2 of cleared lysate sample on a 1 agarose gel and compare to the eluted plasmid DNA as shown in Figure 2 If you find that you ha
8. d copy number Plasmid and cosmids vary in copy number depending on the origin of replication they contain their size and the size of insert Protocols for both high and low copy number plasmids are provided For more details visit our plasmid resource page at www qiagen com goto plasmidinfo and click on the link General Considerations for Optimal Results Host strains The strain used to propagate a plasmid can have a substantial influence on quality of the purified DNA Host strains such as DH5 a and C600 yield high quality DNA with QIAGEN protocols The slower growing strain XL1 Blue also yields DNA of very high quality Strain HB101 and its derivatives such as TG1 and the JM100 series contain large amounts of carbohydrates that are released during lysis and can inhibit enzyme activities if not completely removed In addition some strains such as JM101 JM110 and HB101 have high levels of endonuclease activity and yield DNA of lower quality If the quality of purified DNA is not as expected a change of host strain should be considered If difficulty is encountered with strains such as TG1 and Top10F we recommend reducing the amount of culture volume to improve the ratio of biomass to lysis buffers for optimized lysis conditions D CompactPrep Plasmid Purification Handbook 04 2012 9 Table 1 Origins of replication and copy numbers of various plasmids and cosmids Origin of Copy DNA constr
9. e used for elution Note TE buffer contains EDTA which may inhibit downstream enzymatic or sequencing reactions Note Store DNA at 20 C when eluted with water as DNA may degrade in the absence of buffering and chelating agents 18 CompactPrep Plasmid Purification Handbook 04 2012 Determination of yield To determine the yield DNA concentration should be determined by both UV spectrophotometry at 260 nm and quantitative analysis on an agarose gel To ensure accuracy make sure the absorbance readings fall into the linear range of your method e g between 0 1 and 1 0 for spectrophotometric OD Agarose gel analysis We recommend removing and saving an aliquot of the cleared lysate If the plasmid DNA is of low yield or quality the sample and eluate can be analyzed by agarose gel electrophoresis to determine the stage of the purification procedure where the problem occurred see page 31 CompactPrep Plasmid Purification Handbook 04 2012 19 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 qgiagen 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
10. format at www giagen com ts msds asp where you can find view and print the MSDS for each QIAGEN kit and kit component 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 Quality Control In accordance with QIAGEN s ISO certified Quality Management System each lot of CompactPrep Plasmid Kit is tested against predetermined specifications to ensure consistent product quality CompactPrep Plasmid Purification Handbook 04 2012 5 Introduction QIAGEN CompactPrep Plasmid Kits provide a novel method for very fast 20 minutes large scale plasmid preparation without the need for large volume centrifuges The procedure is based on a novel non chaotropic binding chemistry reducing the total preparation volume to miniprep scale All protocol steps after cell harvest can be performed at the bench using QIAfilter cartridges a vacuum manifold and a standard microcentrifuge CompactPrep Plasmid Kits do not contain QIAfilter cartridges and require lysate clearing by centrifugation The unique kit chemistry and design of the CompactPrep column ensure large scale plasmid prep using a microspin column format CompactPrep Plasmid Kits provide molecular biology grade DNA highly suited for routine applications such as sequencing enzymatic modification cloning and transfection into robust cell lines such a
11. hieved by plating serial dilutions of a culture onto LB agar plates in the absence of antibiotics The counted colonies are used to calculate the number of cells per milliliter which is then set in relation to the measured OD values Analytical gel analysis The success of the plasmid purification procedure can be monitored on an analytical gel see Figure 2 page 31 We recommend removing and saving an aliquot of the cleared lysate If the plasmid DNA is of low yield or quality the sample and eluate can be analyzed by agarose gel electrophoresis to determine the stage of the purification where the problem occurred see page 31 CompactPrep Plasmid Purification Handbook 04 2012 11 Convenient stopping points in protocols For all protocols the purification procedure can be stopped and continued later by freezing the cell pellets obtained by centrifugation The frozen cell pellets can be stored at 20 for several weeks These stopping points are indicated by the symbol Using LyseBlue reagent 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
12. inoculate a starter culture of 2 5 ml LB medium containing the appropriate selective antibiotic Incubate for approximately 8 h at 37 with vigorous shaking approximately 300 rpm Use a tube or flask with a volume of at least 4 times the volume of the culture 2 Dilute the starter culture 1 500 to 1 1000 into selective LB medium For high copy plasmids inoculate 25 ml or A 100 ml medium For low copy plasmids inoculate 50 ml or A 250 ml medium Grow at 37 for 12 16 hours with vigorous shaking approximately 300 rpm Do not increase the culture volume as this will lead to a decrease in DNA yield and quality Use a flask or vessel with a volume of at least 4 times the volume of the culture The culture should reach a cell density of approximately 3 4 x 10 cells ml which typically corresponds to a pellet wet weight of roughly 3 g liter 3 Harvest the bacterial cells by centrifugation at 6000 x g for 15 min at 4 C If you wish to stop the protocol and continue later freeze the cell pellets at 20 4 Resuspend the bacterial pellet in 2 ml or A 5 ml Buffer For efficient lysis it is important to use a vessel that is large enough to allow complete mixing of the lysis buffers Ensure that RNase A has been added to Buffer P1 The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain If LyseBlue reagent has been added to Buffer P1 vigorously shake
13. lution to warm up and become clear before proceeding with the next step Alternatively allow cleared lysate to warm to room temperature before adding Buffer BB 11 Switch on vacuum source to draw the solution through the CompactPrep column and then switch off vacuum source 12 To wash the DNA using a microcentrifuge proceed with step 12a Alternatively to wash the DNA using a vacuum manifold proceed to step 12b 12a To wash the DNA using a microcentrifuge Discard the tube extenders and place the CompactPrep column into a 2 ml collection tube provided Wash the CompactPrep column by adding 0 7 ml Buffer PE and centrifuging for 30 60 s Discard the flow through and centrifuge for an additional 1 min to remove residual wash buffer 12b To wash the DNA using a vacuum manifold Discard the tube extenders Add 0 7 ml Buffer PE to the column and switch on the vacuum manifold To completely remove residual wash buffer continue to apply vacuum for a further 10 min after the solution has been drawn through Note The residual wash buffer can be removed by centrifugation of the CompactPrep column for 1 min in a microcentrifuge 13 Place the CompactPrep column in a clean 1 5 ml microcentrifuge tube To elute DNA add 100 pl or A 200 ul of Buffer EB 10 mM Tris Cl pH 8 5 or water to the center of the CompactPrep column let stand for 1 min then centrifuge for 1 min Water or buffers commonly used to dissolve DNA e g TE may also b
14. mic DNA Multimeric plasmid DNA can easily be distinguished from genomic DNA by a simple restriction digestion linearization of a plasmid sample displaying multimeric bands will yield a single defined band with the size of the linearized plasmid monomer see lane 3 Lane 3 Linearized form of plasmid pTZ19 after restriction digestion with EcoRI Lane 4 Sample contaminated with bacterial chromosomal DNA which may be observed if the lysate is treated too vigorously e g vortexing during incubation steps with Buffer P2 or Buffer P3 Genomic DNA contamination can easily be identified by digestion of the sample with EcoRI A smear is observed in contrast to the linear band seen after digestion of multimeric plasmid forms Lane 5 EcoRI digestion of a sample contaminated with bacterial genomic DNA which gives a smear above the plasmid DNA M2 Lambda DNA digested with Hindlll AXI CompactPrep Plasmid Purification Handbook 04 2012 23 Ordering Information Product Contents Cat no CompactPrep Plasmid 25 CompactPrep Midi Columns 12843 Midi Kit 25 Extender tubes Reagents Buffers CompactPrep Plasmid 25 CompactPrep Maxi Columns 12863 Maxi Kit 25 Extender tubes Reagents Buffers Accessories QlAvac 24 Plus Vacuum manifold for processing 19413 1 24 spin columns QlAvac 24 Plus Vacuum manifold Luer Plugs Quick Couplings CompactPrep Plasmid Kits require use of a vacuum device for operation e g QlAvac 24 Plu
15. product yield LyseBlue can be added to the resuspension buffer Buffer P1 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 ul 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 homogeneously 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 homogeneously colored suspension is achieved Upon addition of neutralization buffer Buffer S3 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 12 CompactPrep Plasmid Purification Handbook 04 2012 Centrifugation All microcentrifugation steps are carried out at 10 000 x g app
16. rification Handbook 04 2012 CompactPrep PlasmidKit Procedure Pelleted bacteria Alkaline lysate i Clear lysate Centrifuge Add binding buffer Pure plasmid plasmid DNA CompactPrep Plasmid Purification Handbook 04 2012 7 Equipment and Reagents to Be Supplied by User When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier Standard microbiological equipment for growing and harvesting bacteria e g inoculating loop culture tubes and flasks 37 C shaking incubator and centrifuge with rotor and tubes or bottles for harvesting cells 96 100 ethanol Microcentrifuge Vacuum manifold e g QlAvac 24 Plus cat 19413 Optional Water or TE buffer for elution Refrigerated centrifuge capable of 220 000 x g with rotor for the appropriate centrifuge tubes or bottles 8 CompactPrep Plasmid Purification Handbook 04 2012 Important Notes Please take a few moments to read this handbook carefully before beginning the DNA preparation If QIAGEN plasmid purification kits are new to you please visit our plasmid resource page at www giagen com goto plasmidinfo and click on the link General Considerations for Optimal Results Also be sure to read and follow the appropriate detailed protocol Plasmid cosmi
17. roximately 13 000 rpm in a conventional tabletop microcentrifuge CompactPrep Plasmid Kits require a refrigerated centrifuge capable of 7 20 000 x g and a rotor for the appropriate centrifuge tubes or bottles for lysate clearing Vacuum manifolds Use of a vacuum manifold is required to draw the DNA solution into the CompactPrep column A waste disposal vessel allowing sufficient volume for the amount of preps run should be attached to the vacuum manifold QlAvac 24 Plus vacuum manifold M Remove the collection tube from the CompactPrep column Do not discard Insert up to 24 CompactPrep Midi or Maxi columns into the luer extensions of the QlAvac 24 Plus Attach a tube extender 10 ml to each CompactPrep Midi column and a tube extender 20 ml to each CompactPrep Maxi column Close unused positions of the manifold with luer caps and connect the QlAvac 24 Plus to a vacuum source See Figure 1 page 18 E Optional VacValves can be used to handle multiple samples with different flow rates Closing VacValves after liquid has been drawn through the first columns ensures that the vacuum pressure remains constant and stable for the remaining samples NI CompactPrep Plasmid Purification Handbook 04 2012 13 Set up of CompactPrep Columns and Tube Extender on a GlAvac 24 Plus Figure 1 Setting up the QlAvac 24 Plus with CompactPrep columns using VacValves 1 QlAvac 24 Plus vacuum manifold 4 CompactPrep column 2 Luer slo
18. s cat no 19413 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 giagen com or can be requested from QIAGEN Technical Services or your local distributor LLELLLLLLZUNI 24 CompactPrep Plasmid Purification Handbook 04 2012 Notes CompactPrep Plasmid Purification Handbook 04 2012 25 Notes 26 CompactPrep Plasmid Purification Handbook 04 2012 Trademarks QIAGEN CompactPrep LyseBlue QIAGEN Group DH5 Life Technologies Inc pBluescript Agilent Technologies Inc pGEM Promega Limited License Agreement for CompactPrep Plasmid 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 the protocols provided with the product this handbook and additional protocols available at www giagen com Some of these additional protocols have been provided by QIAGEN users for QIAGEN users These protocols have not been thoroughly tested
19. s HeLa COS 7 CHO HEK 293 and NIH 3T3 A higher DNA quality may be required for transfection into primary or sensitive cell lines QIAGEN offers the most comprehensive portfolio of tailored plasmid purification kits for any scale throughput or downstream application Select the optimum kit for your requirements by visiting our online selection guide at www giagen com products plasmid selectionguide Principle and procedure The CompactPrep Plasmid Kit protocol is based on a modified alkaline lysis procedure A novel binding buffer Buffer BB is added to the cleared lysate to optimize plasmid DNA binding under non chaotropic conditions to the membrane of the CompactPrep column The combination of CompactPrep columns with the newly developed binding chemistry uniquely allows large scale yields of plasmid DNA to be obtained using a microspin column format A vacuum manifold e g 24 Plus cat no 19413 is used to draw the cleared lysate and subsequent wash buffer through the CompactPrep column DNA is eluted in low volumes 100 ul Midi 200 ul Maxi of elution buffer Buffer EB by centrifugation using a microcentrifuge The highly concentrated DNA typically gt 1 ug Ll is ready for immediate use without the need for further alcohol precipitation LyseBlue reagent Use of LyseBlue is optional and is not required to successfully perform plasmid preparations See Using LyseBlue reagent on page 16 6 CompactPrep Plasmid Pu
20. smid DNA Important points before starting Text marked with a denotes values for the CompactPrep Plasmid Midi Kit text marked with a denotes values for the CompactPrep Plasmid Maxi Kit Optional remove samples at the indicated steps to monitor the procedure on an analytical gel see appendix page 31 Things to do before starting Add the provided RNase A solution to Buffer P1 before use Use 1 vial RNase A centrifuge briefly before use per bottle Buffer P1 for a final concentration of 100 ug ml Add ethanol 96 10096 to Buffer PE before use see bottle label for volume Check Buffer P2 and Buffer BB for precipitation due to low storage temperature and if necessary dissolve by warming to 37 C Close the bottle containing Buffer P2 immediately after use to avoid acidification of Buffer P2 from in the air CompactPrep Plasmid Purification Handbook 04 2012 15 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 ul 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 16 Procedure 1 Pick a single colony from a freshly streaked selective plate and
21. t of the QlAvac 24 Plus 5 Tube extender 3 VacValve optional 6 Luer slot closed with luer plug Must be purchased separately Other vacuum manifolds Follow the supplier s instructions Insert each CompactPrep column into a luer connector 14 CompactPrep Plasmid Purification Handbook 04 2012 Protocol Purification of Plasmid DNA using CompactPrep Plasmid Kits This protocol is designed for the preparation of up to 200 ug of high copy plasmid DNA using the CompactPrep Plasmid Midi Kit or up to A 750 ug using the CompactPrep Plasmid Maxi Kit In this protocol centrifugation is used to clear bacterial lysates Table 4 Maximum recommended culture volumes CompactPrep Plasmid CompactPrep Plasmid Midi Maxi High copy plasmid 25 100 Low copy plasmid t 50 250 For high copy plasmids expected yields are 100 200 ug for the CompactPrep Plasmid Midi Kit and 300 750 for the CompactPrep Plasmid Maxi Kit For low copy plasmids expected yields are 30 100 for the CompactPrep Plasmid Midi Kit and A 50 250 ug for the CompactPrep Plasmid Maxi Kit using these culture volumes t Low copy plasmids can be efficiently purified using CompactPrep Plasmid Kits however growing bacteria to high cell density or in rich media e g Terrific Broth TB or 2x YT may lead to a reduction in plasmid purity This is due to the increased ratio of contaminants e g RNA proteins and polysaccharides compared to pla
22. the buffer bottle before use to ensure LyseBlue particles are completely resuspended The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain 16 CompactPrep Plasmid Purification Handbook 04 2012 5 Add Bi 2 ml A 5 ml Buffer P2 mix thoroughly by vigorously inverting the sealed tube 4 6 times and incubate at room temperature 15 25 for 3 min The lysate should appear viscous Do not allow the lysis reaction to proceed for more than 5 min After use the bottle containing Buffer P2 should be closed immediately to avoid acidification from in the air If LyseBlue has been added to Buffer P1 the cell suspension will turn blue after addition 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 6 Add Bi 2 ml or A 5 ml Buffer S3 to the lysate and mix immediately by vigorously inverting 4 6 times Proceed directly to step 7 Do not incubate the lysate on ice After addition of Buffer S3 a fluffy white precipitate containing genomic DNA proteins cell debris and KDS becomes visible If the mixture still appears viscous and brownish more mixing is required to completely neutralize the solution If LyseBlue reagent has been used the suspension should be mixed until all
23. ube Extenders 20 ml 25 Collection Tubes 2 ml 25 25 Buffer P1 2x50ml 3 x 50 ml Buffer P2 4 x 20 ml 150 ml Buffer S3 70 ml 2x70 ml Buffer BB 70 ml 2x70 ml Buffer PE concentrate 6 ml 6 ml Buffer EB 15 ml 15 ml RNase 2x5 mg 3x5 mg LyseBlue 2x 50 ul 3 x 50 ul Quick Start Protocol 1 Provided as a 10 mg ml solution Storage CompactPrep Plasmid Kits should be stored dry at room temperature 15 25 Kits can be stored for up to 2 years without showing any reduction in performance and quality After adding RNase A Buffer P1 should be stored at 2 8 C and is stable for 6 months Other buffers and RNase A stock solution can be stored for 2 years at room temperature Intended Use CompactPrep Plasmid 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 4 CompactPrep Plasmid Purification Handbook 04 2012 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
24. uct replication number Classification Plasmids pUC vectors pMB 1 500 700 High copy pBluescript vectors ColE1 300 500 High copy pGEM vectors 1 300 400 High copy pTZ vectors pMB 1 gt 1000 High copy pBR322 and 1 15 20 Low copy derivatives pACYC and P15A 10 12 Low copy derivatives pSC101 and pSC101 5 Very low copy derivatives 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 plasmids listed here contain mutated versions of this origin Culture media QIAGEN plasmid purification protocols are optimized for use with cultures grown in Luria Bertani LB medium to a cell density of approximately 3 4 x 107 cells ml which typically corresponds to a pellet wet weight of approximately 3 g liter medium Please note that a number of slightly different LB culture broths containing different concentrations of NaCl are commonly used We recommend growing cultures in LB medium containing 10 g NaCl per liter Table 2 page 15 to obtain the highest plasmid yields Rich media are not recommended for plasmid preparation with CompactPrep columns If rich media must be used growth time must be optimized and culture volumes reduced For more details visit our plasmid resource page at www giagen com goto plasmidinfo and click on the link General Considerations for Optimal Results
25. ve a problem with a particular step of the protocol turn to the hints in the relevant section of the troubleshooting guide on pages 29 30 If the problem remains unresolved or if you have any further questions please call QIAGEN Technical Service When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier A NAA yAO L O OOOLI OeAIOL OOO OAOCACAA AAAAAAAAAOAAAOeEEDAA AAAOOAOOAAAAAAADOeE LO DLO LAOLLL IVUL O q LLL AAOAaaAm l 22 CompactPrep Plasmid Purification Handbook 04 2012 L Cleared lysate containing supercoiled and open circular plasmid DNA and degraded RNA E The eluate containing pure plasmid DNA with no other contaminating nucleic acids 1 kb ladder 1 2 3 4 6 10 kb Lanes 1 5 illustrate some atypical results that may be observed in some preparations depending on plasmid type and host strain Lane 1 Supercoiled lower band and open circular form upper band of the high copy plasmid pUC18 with an additional band of denatured supercoiled DNA migrating just below the supercoiled form This form may result from prolonged alkaline lysis with Buffer P2 and is resistant to restriction digestion Lane 2 Multimeric forms of supercoiled plasmid DNA pTZ19 which may be observed with some host strains and should not be mistaken for geno
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