QIAprep 96 Plus Miniprep Handbook
Contents
1. Add ethanol 96 100 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 CO in the air Assemble the QlAvac 96 as described on page 12 The vacuum should be regulated to 300 mbar before beginning the procedure QlAprep 96 Plus Miniprep Handbook 04 2012 15 Procedure 1 Resuspend pelleted bacteria in 300 pl Buffer P1 IMPORTANT Ensure that RNase A has been added to Buffer P1 Note Resuspend the bacterial pellet completely by vortexing or pipetting up and down until no cell clumps remain If frozen cells are used make sure that the cells are completely thawed also in the centre of the Flat Bottom Block If cells were not harvested in a Flat Bottom Block transfer resuspended bacteria into the Flat Bottom Block 2 Add 300 pl Buffer P2 to each well Dry the top of the Flat Bottom Block thoroughly with a paper towel and seal the block with the tape provided Gently invert the block upside down 6 8 times to mix and incubate at room temperature 15 25 for 5 min Note 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 clear2. Note Unused wells of the Plasmid Plus 96 plate should be sealed with tape 8 Apply vacuum until all samples have passed through Note After the liquid has been drawn through all wells switch off the vacuum source and ventilate the QlAvac 96 slowly 9 To wash the DNA add 900 ul Buffer PE to each well of the Plasmid Plus 96 plate Apply vacuum until all samples have passed through Note After the liquid has been drawn through all wells switch off the vacuum source and ventilate the QlAvac 96 slowly 10 To dry the membranes of the plate use the QlAvac 96 with the centrifuge or a vacuum manifold Note This step removes residual Buffer PE from the membrane by airflow going through the wells If using a centrifuge place the Plasmid Plus 96 plate onto an S Block or elution microtube rack with elution microtubes and centrifuge at 6000 x g for 10 min IMPORTANT S blocks or elution microtubes required here are not provided with the kit If using vacuum empty the waste tray and put it back into the QlAvac 96 and apply maximum vacuum for 10 min Switch off vacuum and ventilate the QlAvac 96 slowly Lift the top plate from the base but not the Plasmid Plus plate 96 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 Plasmid Plus 96 plate with clean absorbent paper Buffer PE removal is only effective when maximum vacuum is used i e turn off va
3. IMPORTANT Do not allow the lysis reaction to proceed for more than 5 min 3 Remove the tape from the block Add 300 pl Buffer S3 to each well Dry the top of the Flat Bottom Block thoroughly with a paper towel and seal the block with new tape Gently invert the block upside down 6 8 times to mix Note To avoid localized precipitation mix the samples gently but thoroughly immediately after addition of Buffer S3 The solutions should become cloudy 4 Remove the tape from the block Transfer the lysates from step 3 into the wells of the TurboFilter 96 plate on the assembled QlAvac 96 Apply vacuum 300 mbar until all samples have passed through the wells of the TurboFilter 96 plate into the S Block Note Unused wells of the TurboFilter 96 plate should be sealed with tape 5 After all liquid has been drawn through the TurboFilter 96 plate switch off the vacuum source and ventilate the QlAvac 96 slowly Note Discard the TurboFilter 96 plate after this step 6 Add 300 ul Buffer BB to the cleared lysate in each well of the S Block Dry the top of the S Block thoroughly with a paper towel and seal the block with a new tape Invert the block upside down 1 3 times to mix 16 QlAprep 96 Plus Miniprep Handbook 04 2012 7 Place a waste tray into the base of the QlAvac 96 Place the QlAvac 96 top plate over the base Place the Plasmid Plus 96 plate in the top plate Transfer the lysates from the S Block to the Plasmid Plus 96 plate
4. in some preparations depending on plasmid type and host strain 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 5055 available from the product supplier QlAprep 96 Plus Miniprep Handbook 04 2012 25 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 genomic 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 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 afte
5. 96 Plus Miniprep Handbook 04 2012 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 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 QlAprep 96 Plus Miniprep protocol 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 It is best to assess the cell density of the culture and if it is too high 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 achieved 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 Assembly of the QlAvac 96 vacuum manifold The QlAvac 96 must be assembled before starting the QlAprep 96 Plu
6. April 2012 QlAprep 96 Plus Miniprep Handbook QlAprep 96 Plus Miniprep Kit QlAprep 96 Plus BioRobot Kit For 96 well preparation of up to 50 ug of high quality 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 9 Important Notes 10 Assembly of the QlAvac 96 vacuum manifold 1 Cell cultivation in a 96 well Flat Bottom Block 12 Cell cultivation in 24 well block 12 Protocol Plasmid DNA Purification using the QIAprep 96 Plus Miniprep Kit 15 Use of a centrifuge for the QlAprep 96 Plus Miniprep procedure 18 Troubleshooting Guide 21 Appendix A Setup of the QlAvac 96 23 Appendix B Agarose Gel Analysis of the Purification Procedure 25 Ordering Informat
7. GEN 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 Japan t North America UK Rest of World 28 QlAprep 96 Plus Miniprep Handbook 04 2012 Notes QlAprep 96 Plus Miniprep Handbook 04 2012 29 Notes 30 QlAprep 96 Plus Miniprep Handbook 04 2012 Trademarks QIAGEN QlAprep BioRobot TurboFilter QIAGEN Group DH5 Life Technologies Inc Heraeus Heraeus Holding GmbH Limited License Agreement for QlAprep 96 Plus Miniprep and BioRobot 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 or optimized by QIAGEN QIAGEN neither guarantees them nor warrants that they do not infringe the rights of third parties Other than expressly stated li
8. Vacuum Manifold Figure 3 Components of the QlAvac 96 vacuum manifold QlAvac base which holds a waste tray S Block or Elution Microtube Adapter Waste tray QlAvac 96 top plate Elution Microtube Rack with Elution Microtubes 96 well plate i e TurboFilter 96 or Plasmid Plus 96 Plate Elution Microtube Adapter not included with the QlAvac 96 please contact QIAGEN Technical Services WN 24 QlAprep 96 Plus Miniprep Handbook 04 2012 Appendix B 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 a 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 pl TE buffer pH 8 0 Agarose gel analysis Run 2 ul of cleared lysate sample on a 1 agarose gel and compare to the eluted plasmid DNA as shown in Figure 4 1 2 3 4 5 M2 Figure 4 Agarose gel analysis of the plasmid purification procedure Lanes 1 5 illustrate some atypical results that may be observed
9. acterial cells in the block by centrifugation for 5 min at 2100 x g in a centrifuge with a rotor for a 96 well adapter e g QIAGEN s Centrifuge 4 16K 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 remaining droplets of medium IMPORTANT Ensure that the buckets on the rotor have sufficient clearance to accommodate the 2 ml Flat Bottom Blocks before starting the centrifuge Cell cultivation in a 24 well block To obtain high plasmid yields of up to 50 a 5 ml LB culture volume per well is used for plasmid preparation E coli cultures can be grown and harvested in 24 well blocks We do not recommend rich media here as the higher amount of biomass may lead to clogging of the TurboFilter 96 plate To ease growth of E coli cultures in parallel cultures can be grown and harvested in 24 well blocks e g QIAGEN s 24 Well Blocks RB cat no 19583 QlAprep 96 Plus Miniprep Handbook 04 2012 Procedure 1 Fill each well of a 24 well block with 5 ml of LB medium containing the appropriate selective agent 2 Inoculate each well from a single bacterial colony or pre culture Carefully seal the block with an AirPore tape sheet cat no 19571 4 Incubate the cultures
10. cal yields of plasmid DNA from different volumes of E coli culture E coli culture volume LB medium Typical DNA yield 1 25 ml Up to 15 ug 2 5 ml Up to 30 ug 5 ml Up to 50 ug When using a 5 ml culture the volumes of the lysis buffers may be slightly increased Use 350 ul Buffer P1 350 ul Buffer P2 350 ul Buffer S3 and 300 ul Buffer BB If high DNA yields are expected the elution volume may be increased to 100 120 ul QlAprep 96 Plus Miniprep Handbook 04 2012 7 QlAprep 96 Plus Miniprep Procedure Plasmid Plus 96 plate High quality plasmid DNA 8 QlAprep 96 Plus Miniprep Handbook 04 2012 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 and 37 C shaking incubator M Centrifuge with rotor for 96 well blocks We recommend QIAGEN s Centrifuge 4 16 cat no 80310 for room temperature centrifugation and for refrigerated centrifugation we recommend QIAGEN s Centrifuge 4 16K cat no 81410 96 100 ethanol Vacuum pump e g QIAGEN Vacuum Pump no 84010 QlAvac 96 cat no 19504 Elution Microtube Adapter available from QIAGEN Technical S
11. censes 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 GO BND 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 2010 2012 QIAGEN all rights reserved www qiagen com Australia techservice au qiagen com Austria techservice at qiagen com Belgium techservice bnl qiagen com Brazil suportetecnico brasil qiagen com Canada techservice ca qiagen com China techservice cn qiagen 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 qiagen com Ireland techservice uk qiagen com Italy techservice it qiagen com Japan techservic
12. cuum regulator or leakage valves if they are used QlAprep 96 Plus Miniprep Handbook 04 2012 17 11 To elute the DNA from the plate use a centrifuge or the QlAvac 96 Note Ensure that Buffer EB is dispensed directly onto the center of the Plasmid Plus 96 plate membrane for optimal elution of DNA Average eluate volume is 65 ul from an elution buffer volume of 80 ul If using a centrifuge M the Plasmid Plus 96 plate onto a new elution microtube rack containing elution microtubes Add 80 ul of Buffer EB to the centre of each well of the Plasmid Plus 96 plate M Let it stand for 3 min and then centrifuge at 6000 x g for 1 min If using the QlAvac 96 M Replace the waste tray with the Elution Microtube Adapter Alternatively if no Elution Microtube Adapter is available an empty 96 well microplate may be used M Place the elution microtube rack containing elution microtubes onto the adapter M Place the top plate back on the base making sure that the Plasmid Plus 96 plate is seated securely If a microplate is used instead of the Elution Microtube Adapter please ensure that the nozzles of the Plasmid Plus 96 plate extend into the elution microtubes Add 80 ul of Buffer EB to the centre of each well of the Plasmid Plus 96 plate M Let it stand for 3 min and then apply maximum vacuum for 1 min M Switch off the vacuum source and ventilate the QlAvac 96 slowly Use of a centrifuge for the QlAprep 96 Plus M
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14. ervices for QlAvac 96 for the elution step using the QlAvac 96 Alternatively an empty microplate may be used to adjust the height of the elution microtubes or the elution step could be performed in a suitable centrifuge where no adapter is necessary M If the drying step is performed in a suitable centrifuge optional additional S Blocks Square Well Blocks or elution microtubes are required M If the complete procedure is performed using a centrifuge additional S Blocks are required QlAprep 96 Plus Miniprep Handbook 04 2012 9 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 qiagen com goto plasmidinfo and click the link General Considerations for Optimal Results Also be sure to read and follow the appropriate detailed protocol Plasmid copy number Plasmid and cosmids vary in copy number depending on the origin of replication they contain their size and the size of insert For more details visit our plasmid resource page at www qiagen com goto plasmidinfo and click 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 DH1 DH5 a and C600 yield high quality DNA with QIAGEN protocols The slower growing strain XL1 Bl
15. for 16 24 h at 37 C with vigorous but appropriate shaking 5 Harvest the bacterial cells in the block by centrifugation for 5 min at 2100 x g in a centrifuge with a rotor for a 96 well adapter e g QIAGEN s Centrifuge 4 16K or Heraeus Minifuge GL preferably at 4 10 C The block should be covered with adhesive tape during centrifugation Remove media by carefully inverting the block Proceed as described in the section Transfer from 24 well format to 96 well format To remove the media peel off the tape and quickly invert the block over a waste container Tap the inverted block carefully on a paper towel to remove any remaining droplets of medium Alternatively the cultures may be harvested using a Flat Bottom Block where aliquots of each of the 5 ml cultures are collected per well by centrifugation with subsequent removal of medium IMPORTANT Ensure that the buckets on the rotor have sufficient clearance to accommodate the 24 well blocks before starting the centrifuge QlAprep 96 Plus Miniprep Handbook 04 2012 13 Transfer from 24 well format to 96 well format To transfer from 24 well format to 96 well format the samples may be transferred to a 96 well flat bottom block after resuspension of pelleted bacteria with Buffer P1 step 1 of the purification protocol Alternatively steps 1 3 of the purification protocol can be performed within the wells of the 24 well blocks For subsequent lysate clearing the lysa
16. he Plasmid Plus 96 plate on top of an elution microtube rack or a clean S Block IMPORTANT S Blocks required in this step are not provided with the kit 14 Centrifuge for 10 min at 6000 x g Elution 15 Place the Plasmid Plus 96 plate on top of a new elution microtube rack containing elution microtubes 16 Add elution buffer Buffer EB as described in the protocol page 18 17 Load the assembled components Plasmid Plus 96 plate on the elution microtubes into the rotor 18 Centrifuge for 1 min at 6000 x g QlAprep 96 Plus Miniprep Handbook 04 2012 19 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 For reliable spectrophotometric DNA quantification readings should lie between 0 1 and 1 0 Agarose gel analysis We recommend removing and saving an aliquot of the cleared lysate step 5 If the plasmid DNA is of low yield or quality the sample and eluate can be analyzed by agarose gel electrophoresis to determine at the stage of the purification procedure where the problem occurred See Appendix B for more information oo 20 QlAprep 96 Plus Miniprep Handbook 04 2012 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 FAQLi
17. ide highly pure DNA suitable for transfection into robust cell lines for preparation of short hairpin vectors sh vectors as well as for routine applications such as enzymatic modifications cloning restriction digestion and in vitro transcription translation Principle and procedure The QlAprep 96 Plus Miniprep protocol is based on a modified alkaline lysis procedure After neutralization lysates are cleared by using a TurboFilter 96 plate A nonchaotropic binding buffer Buffer BB is added to the cleared lysate to optimize plasmid DNA binding to the membrane of the Plasmid Plus 96 plate The unique binding buffer provides very specific binding conditions leading to DNA quality that is comparable to anion exchange preps Due to the proprietary binding chemistry up to 50 ug plasmid DNA per well can be obtained from a 5 ml E coli culture The DNA yield depends on the plasmid copy number and the amount of E coli culture used The procedure may be automated using QlAprep Plus 96 BioRobot Kit 4 cat no 962241 on the BioRobot Universal System The QlAvac 96 is used for lysate clearing using the TurboFilter 96 plate and DNA binding and wash steps using the Plasmid Plus 96 plate Drying and elution steps may be performed either on the QlAvac 96 or using a compatible centrifuge Alternatively the complete procedure can be performed by using a compatible centrifuge SSeS 6 QlAprep 96 Plus Miniprep Handbook 04 2012 Table 1 Typi
18. iniprep procedure The QlAprep 96 Plus Miniprep procedure may be performed using a centrifuge that is suitable for 96 well blocks Before starting check if the centrifuge can accommodate 96 well plates and the S Block Replace the vacuum steps with the centrifugation steps as described below For further details individual steps of the purification procedure refer to the protocol on pages 17 18 Lysate clearing with the TurboFilter 96 plate 1 After step 3 of the protocol place the TurboFilter 96 plate on top of an S Block 2 Transfer the lysates onto the TurboFilter 96 plate 18 QlAprep 96 Plus Miniprep Handbook 04 2012 3 Load the assembled components TurboFilter 96 plate on the S Block into the rotor 4 Centrifuge for 3 min at 3000 x g Binding 5 After step 6 of the protocol place the Plasmid Plus 96 plate on top of an S Block IMPORTANT S Blocks required in this step are not provided with the kit 6 Transfer the lysates onto the Plasmid Plus 96 plate 7 Load the assembled components Plasmid Plus 96 plate on the S Block into the rotor 8 Centrifuge for 1 min at 160 xg Wash steps 9 Place the Plasmid Plus 96 plate on top of an S Block IMPORTANT S Blocks required in this step are not provided with the kit 10 Add the respective wash buffers 11 Load the assembled components Plasmid Plus 96 plate on the S Block into the rotor 12 Centrifuge for 1 min at 160 xg Drying 13 Place t
19. ion 27 amp QlAprep 96 Plus Miniprep Handbook 04 2012 3 Kit Contents QlAprep 96 Plus QlAprep 96 Plus Kit Miniprep Kit 4 BioRobot Kit 4 Catalog no 27291 962241 Number of preps 4 4 Buffer 150 ml 2 x 250 ml Buffer P2 140 ml 2 x 250 ml Buffer S3 160 ml 2 x 160 ml Buffer BB 160 ml 2 x 160 ml Buffer PE concentrate 100 ml 2x 100 ml Buffer EB 55 ml 2x 55 ml RNase A 100 mg ml 15 mg 2x25 mg TurboFilter 96 Plates 4 4 Plasmid Plus 96 Plates 4 4 Tape Pad Flat Bottom Blocks S Blocks 4 4 Elution Microtubes racked 96 4 4 Quick Start Protocol Storage QlAprep 96 Plus Miniprep and BioRobot Kits should be stored dry at room temperature 15 25 C Kits can be stored for up to 1 year 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 QlAprep 96 Plus Miniprep and BioRobot Kits are intended for molecular biology applications These products are not intended for the diagnosis prevention or treatment of a disease 4 QlAprep 96 Plus Miniprep Handbook 04 2012 All due care and attention should be exercised in the handling of the products We recommend all users of QIAGEN products
20. or Vacuum Pump e g Vacuum Pump cat no 84010 USA and Canada 84000 Japan or 84020 rest of world M Always store QlAvac manifolds clean and dry To clean simply rinse all components 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 M Always place the QlAvac manifold on a secure bench top or work area If dropped the manifold may crack M The components of QlAvac manifolds are not resistant to ethanol methanol or other organic solvents Table 3 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 To ensure consistent performance do not apply silicone or vacuum grease to any part of a QlAvac manifold The spring lock on the top plate and the self sealing gasket 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 3 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 QlAprep 96 Plus Miniprep Handbook 04 2012 23 QlAvac 96
21. r 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 Hindi a 26 QlAprep 96 Plus Miniprep Handbook 04 2012 Ordering Information Product QlAprep 96 Plus Miniprep Kit 4 QlAprep 96 Plus BioRobot Kit 4 Contents For 4 x 96 plasmid minipreps TurboFilter 96 Plates Plasmid Plus 96 Plates Buffers Reagents Flat Bottom Blocks S Blocks and Elution Microtubes For 4 x 96 plasmid minipreps TurboFilter 96 Plates Plasmid Plus 96 Plates Buffers Reagents Flat Bottom Blocks S Blocks and Elution Microtubes QIAGEN Plasmid Plus 96 Kits for purification of up to 50 Hg Transfection Grade plasmid DNA in 96 well format QIAGEN Plasmid Plus 96 Miniprep Kit 4 QIAGEN Plasmid Plus 96 BioRobot Kit 4 Accessories QlAvac 96 For 4 x 96 plasmid minipreps TurboFilter 96 Plates Plasmid Plus 96 Plates Buffers Reagents 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 For 4 x 96 plasmid minipreps TurboFilter 96 Plates and Plasmid Plus 96 Plates Buffers Reagents Flat Bottom Blocks S Blocks and Elution Microtubes for use with the BioRobot Universal System Vacuum manifold for processing QIAGEN 96 well plates includes QlAvac 96 Top Plate Base Was
22. rred according to the given numerical scheme 14 QlAprep 96 Plus Miniprep Handbook 04 2012 Protocol Plasmid DNA Purification using the QlAprep 96 Plus Miniprep Kit This protocol is designed for the preparation of up to 50 ug high copy plasmid DNA using the QlAprep 96 Plus Miniprep Kit with a maximum culture volume i e LB medium of 5 ml per well Important points before starting Wear safety glasses when working near a vacuum manifold under pressure Always place the QlAvac 96 manifold on a secure bench top or work area For safety reasons do not use 96 well plates that have been damaged in any way To avoid the possibility of implosion do not use any vessel material that is not designed for use with a vacuum Do not use any material that is cracked or scratched Optional samples can be removed after step 5 of the protocol to monitor the procedure on an analytical gel Switch off the vacuum source between steps to ensure that a consistent even vacuum is applied during vacuum steps When using a 5 ml culture the volumes of the lysis buffers may be slightly increased use 350 ul Buffer P1 350 ul Buffer P2 350 ul Buffer S3 and 300 ul Buffer BB If high DNA yields are expected the elution may be increased to 100 120 ul 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
23. s Miniprep protocol Refer to Appendix A page 23 for further details 1 Place an S Block inside the QlAvac base 2 Place the QlAvac 96 top plate over the base 3 Place the TurboFilter 96 plate into the QlAvac top plate Make sure that the plate is seated securely The S Block should now be positioned under the TurboFilter 96 plate Attach the QlAvac 96 to a vacuum source Seal the TurboFilter 96 plate with tape and apply vacuum Using a vacuum regulator adjust vacuum to 300 mbar Remove the tape NOR QlAprep 96 Plus Miniprep Handbook 04 2012 11 Table 2 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 Cell cultivation in a 96 well Flat Bottom Block 1 2 3 Fill each well of a 96 well Flat Bottom Block with 1 3 ml of growth medium containing the appropriate selective agent Inoculate each well from a single bacterial colony or pre culture Incubate the cultures for 20 24 h at 37 C with vigorous shaking The wells in the block may be protected against spill over by covering the block with an AirPore tape sheet cat no 19571 AirPore microporous tape sheets promote gas exchange during culturing If nonporous tape is used pierce 2 3 holes in the tape with a needle above each well for aeration Harvest the b
24. st 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 Comments and suggestions Low or no yield No DNA in the cleared lysate before loading a Plasmid did not Check that the conditions for optimal growth propagate 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 efficient release of plasmid DNA 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 S3 to achieve homogeneous suspensions c Buffer P2 or BB Redissolve by warming to 37 C precipitated d Cell resuspension Pelleted cells should be completely resuspended incomplete in Buffer P1 Do not add Buffer P2 until a homogeneous suspension is obtained DNA is found in the wash flow through Ethanol omitted from Repeat procedure with correctly prepared wash wash buffer buffer Buffer PE QlAprep 96 Pl
25. te Tray Plate Holder Rack of Collection Microtubes 1 2 ml Cat no 27291 962241 16181 960241 19504 Requires use of QlAvac 96 and Elution Microtube Adapter contact QIAGEN Technical Services or a centrifugation system suitable for 96 well blocks t For use with the BioRobot Universal System Available from QIAGEN Technical Services QlAprep 96 Plus Miniprep Handbook 04 2012 27 Product Contents Cat no BioRobot Universal Robotic workstation computer 9001094 System controlled vacuum pump computer QIAsoft 5 Operating System installation 1 year warranty on parts and labor Centrifuge 4 16 Universal laboratory centrifuge with 81300 brushless motor 81310t 81325 813208 Centrifuge 4 16K Refrigerated universal laboratory 81400 centrifuge with brushless motor 814101 81425 814208 24 Well Blocks 24 24 well blocks with 10 ml round 19583 bottom wells 24 per case 48 Well Blocks 24 48 well blocks with 5 ml wells 24 per 19577 Elution Microtubes Nonsterile polypropylene tubes 19588 24 x 96 0 85 ml maximum capacity less than 0 7 ml storage capacity 0 4 ml elution capacity 2304 in racks of 96 includes cap strips S Blocks 24 96 well blocks with 2 2 ml wells 24 per 19585 case AirPore Tape Sheets Microporous tape sheets for covering 19571 50 96 well blocks 50 sheets per pack For up to date licensing information and product specific disclaimers see the respective QIA
26. tes are directly transferred from the wells of the 24 well blocks to the wells of the TurboFilter 96 plate The following pipetting scheme may be used for transfer of the lysates Transfer from the 24 well block may be performed by using every second channel of an 8 channel pipet M Transfer lysates from the first block to rows 1 6 only to lanes A C and G of the Flat Bottom Block or TurboFilter 96 plate M Transfer lysates from the second block to rows 7 12 only to lanes A E and G of the Flat Bottom Block or TurboFilter 96 plate M Transfer lysates from the third block to rows 1 6 only to lanes B D F and H of the Flat Bottom Block or TurboFilter 96 plate M Transfer lysates from the fourth block to the rows 7 12 only to lanes B D F and H of the Flat Bottom Block or TurboFilter 96 plate Block 1 Block 2 Block 3 Block 4 Figure 1 24 well blocks 1 4 Rows of the TurboFilter 96 plate 1 Je I5 Ia 1 TIo m A 1 1 1 1 1 1 2 2 2 2 2 2 a LB 13 13 13 3 3 4 4 4 4 4 4 1 1 1 1 1 1 2e 2 2 2 2 5 3 13 13 13 13 13 14 4 4 4 4 4 E 1 11 1 1 1 1 2 2 2 2 2 2 F 3 13 13 13 3 3 4 4 4 4 4 4 G 1 1 1 1 1 1 2 2 2 2 2 2 H 3 13 13 13 13 13 4 4 4 4 4 4 Figure 2 Lanes and rows of the 96 well TurboFilter 96 plate to which lysates from the 24 well blocks are transfe
27. 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 qiagen com Support MSDS aspx 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 the QlAprep 96 Plus Miniprep and BioRobot Kits is tested against predetermined specifications to ensure consistent product quality QlAprep 96 Plus Miniprep Handbook 04 2012 5 Introduction QlAprep 96 Plus Miniprep and BioRobot Kits provide a novel method for plasmid preparation The procedure is based on a nonchaotropic binding chemistry Following lysate clearing a simple bind wash elute procedure results in highly concentrated plasmid DNA ready for direct use in subsequent applications The unique kit chemistry and design of the Plasmid Plus 96 plates allow high binding capacities QlAprep 96 Plus Miniprep and BioRobot Kits prov
28. ue also yields DNA of very high quality Strain HB101 and its derivatives such as 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 Culture media QIAGEN plasmid purification protocols are optimized for use with cultures grown in Luria Bertani LB 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 prepared as described on the following page to obtain the highest plasmid yields In general we do not recommend using rich media with our plasmid kits Using rich media might lead to clogging of the filter If this is the case growth time must be optimized and culture volumes reduced For more details visit our plasmid resource page at www qiagen com goto plasmidinfo and click on the link General Considerations for Optimal Results 10 QlAprep
29. us Miniprep Handbook 04 2012 21 Comments and suggestions Low DNA quality Eluate contains residual Ensure that the membrane of the Plasmid Plus 96 ethanol omitted from plate is dried sufficiently using a centrifuge or wash buffer vacuum see step 10 on page 18 of the protocol TurboFilter 96 plate clogs during filtration a Culture volume too Do not exceed the culture volume recommended large in the protocol b Inefficient mixing after Mix well until the solution is cloudy Mix the addition of Buffer S3 samples immediately after addition of Buffer S3 to avoid local precipitation c Mixing too vigorous After addition of Buffer S3 the lysate should be after addition of Buffer mixed immediately but gently Vigorous mixing 3 disrupts the precipitate into tiny particles d Block was agitated Gently mix after addition of Buffer S3 Agitation causes shearing of DNA e The TurboFilter 96 After addition of Buffer S3 the lysate should be plate was not loaded transferred to the TurboFilter 96 plate immediately after immediately addition of Buffer 53 f Vacuum pressure was Ensure that the vacuum generates a vacuum too low pressure of 200 to 600 millibar 150 to 450 mmHg 22 QlAprep 96 Plus Miniprep Handbook 04 2012 Appendix A Setup of the QlAvac 96 Guidelines for QlAvac manifolds The following recommendations should be followed when handling QlAvac manifolds QlAvac manifolds operate with a house vacuum
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