Sample & Assay Technologies BioSprint® 96 DNA
Contents
1. 11 Check that the protective cover is correctly installed it should fit exactly into the body of the BioSprint 96 Slide the door shut to protect samples from contamination See the BioSprint 96 User Manual for safety information 12 Press Start to start sample processing 13 After the samples are processed remove the plates and blocks as instructed by the display of the BioSprint 96 Press Start after removing each plate or block The first item to be removed contains the purified samples Carryover of magnetic particles in eluates will not affect most downstream applications If the risk of magnetic particle carryover needs to be minimized the microplate containing eluates should first be placed in a suitable magnet and the eluates transferred to a clean microplate see the appendix page 50 14 Press Stop after all plates and blocks are removed 15 Discard the used plates blocks and 96 rod cover according to your local safety regulations Note See page 5 for safety information 16 Switch off the BioSprint 96 at the power switch 17 Wipe the worktable and adjacent surfaces using a soft cloth or tissue moistened with distilled water or detergent solution If infectious material is spilt on the worktable clean using 70 ethanol or other disinfectant Note Do not use bleach as disinfectant See page 5 for safety information BioSprint 96 DNA Handbook 06 2012 35 Protocol Purification of DNA from2. M Check that QIAGEN Protease Buffer AW1 Buffer AW2 and RNase free water have been prepared according to the instructions on pages 16 17 M Check that Buffer AL does not contain a white precipitate If necessary incubate Buffer AL for 30 minutes at 37 C with occasional shaking to dissolve precipitate E Ensure that you are familiar with operating the BioSprint 96 Refer to the BioSprint 96 User Manual for operating instructions M 96 rod covers are supplied either as packets of two or as packets of one inserted into an S Block If using a new packet of two store the second 96 rod cover on another plate It is important that the 96 rod cover does not become bent Things to do before starting M Set a shaker incubator with an adapter for S Blocks or 1 5 ml microcentrifuge tubes if lysis is not to be performed in an S Block to 70 C for use in step 5 of the procedure M MagAttract magnetic particles copurify RNA and DNA if both are present in the sample If RNA free DNA is required add RNase A to the sample in step 2 of the procedure The final RNase A concentration should be 2 mg ml e g add 4 ul of a 100 mg ml RNase A solution to a 200 ul sample E Prepare a master mix according to the table on the next page for use in step 7 of the procedure Before adding MagAttract Suspension G ensure that it is fully resuspended Vortex for 3 minutes before using for the first time and for 1 minute before subsequent uses Note Prepa
3. a SSS SSS SS eS ee 22 BioSprint 96 DNA Handbook 06 2012 Protocol Rapid Purification of DNA from Human Whole Blood This protocol is for rapid purification of total genomic and mitochondrial DNA from human whole blood using the BioSprint 96 workstation and the BioSprint 96 DNA Blood Kit Blood samples can be 100 ul or 200 ul This shorter protocol has less manual handling steps than the standard protocol see Protocol Purification of DNA from Blood Using the BioSprint 96 page 18 but yield and purity of the purified DNA may be lower Important points before starting M Check that QIAGEN Protease Buffer AW1 Buffer AW2 and RNase free water have been prepared according to the instructions on pages 16 17 M Check that Buffer AL does not contain a white precipitate If necessary incubate Buffer AL for 30 minutes at 37 C with occasional shaking to dissolve precipitate E This protocol is suitable for human whole blood Blood samples must be in the range of 100 200 ul E Ensure that you are familiar with operating the BioSprint 96 Refer to the BioSprint 96 User Manual for operating instructions M 96 rod covers are supplied either as packets of two or as packets of one inserted into an S Block If using a new packet of two store the second 96 rod cover on another plate It is important that the 96 rod cover does not become bent M In some steps of the procedure one of two choices can be made Choose E if processing 10
4. Handbook 06 2012 19 Table 5 BioSprint 96 worktable setup and reagent volumes Volume per well ul Message when Slot loading Plate block To add m 8 Load 96 well Large Rod microplate 96 Rod Cover MP Cover 7 Load 96 well Buffer AE 100 200 200 Elution microplate MP 6 Load S Block RNase free 500 500 500 Wash 5 water 5 Load S Block Buffer AW2 500 500 500 Wash 4 4 Load S Block Buffer AW2 500 500 500 Wash 3 3 Load S Block Buffer AW1 500 500 500 Wash 2 2 Load S Block Buffer AW1 500 650 800 Wash 1 1 Load S Block Lysatet 325 650 810 Lysate Contains 0 02 v v Tween 20 t Added at steps 2 3 6 and 7 includes volume of QIAGEN Protease sample Buffer AL isopropanol and MagAttract Suspension G Pipet W 10 ul 20 ul or 25 ul QIAGEN Protease into the bottom of a well of an S Block Add W 100 ul 200 ul or 250 pl sample to the QIAGEN Protease Note It is possible to add QIAGEN Protease to samples that have already been dispensed into the S Block In this case it is important to ensure proper mixing after adding QIAGEN Protease Note Record in which wells you load the samples BioSprint 96 DNA Handbook 06 2012 3 Add EM 100 ul 200 ul or 250 pl Buffer AL and seal the S Block with a tape sheet not supplied Mix by pulse vortexing for 10 s To ensure efficient lysis it is essential that the sample and Buffer AL are mixed thoroughly to yield a homogeneous solut
5. M markers Lambda Hindlll 14 BioSprint 96 DNA Handbook 06 2012 M Mouse tail Figure 2 Purification of high quality genomic DNA from mouse tail Mouse tail samples were treated with 180 ul Buffer ATL and 20 ul QIAGEN Proteinase K at 56 C overnight Genomic DNA was purified from the lysed tissue samples using the BioSprint 96 DNA Blood Kit with the BioSprint 96 DNA Tissue protocol DNA was eluted in 200 pl elution buffer Eluates 2 ul from 8 out of 96 samples were visualized by agarose gel electrophoresis M markers 1 kb ladder 0 5 10 15 20 29 30 35 40 Cycle Figure 3 Efficient and sensitive real time PCR Genomic DNA was purified from mouse tail samples after overnight lysis at 56 C with 180 ul Buffer ATL and 20 ul QIAGEN Proteinase K Purification was carried out using the BioSprint 96 DNA Blood Kit with the BioSprint 96 DNA Tissue protocol The c jun gene was amplified using 5 ul purified DNA Amplification reactions 50 ul were carried out on the Rotor Gene system using the QuantiTect Probe PCR Kit with gene specific primers and probe BioSprint 96 DNA Handbook 06 2012 15 Preparing reagents QIAGEN Protease Pipet Protease Solvent which is nuclease free water containing 0 04 w v sodium azide into the vial containing lyophilized QIAGEN Protease as described on the vial label Reconstituted QIAGEN Protease is stable for up to 2 months when stored at 2 8 C Storing reconstituted QIAGEN Protease at
6. Rodent Tails This protocol is for the purification of total genomic and mitochondrial DNA from up to 1 2 cm approximately 25 mg of rodent tail per sample using the BioSprint 96 workstation and the BioSprint 96 DNA Blood Kit Important points before starting E Buffer ATL QIAGEN Proteinase K and tape sheets are required for this protocol See Equipment and Reagents to Be Supplied by User page 9 E Check that Buffer AW1 Buffer AW2 and RNase free water have been prepared according to the instructions on pages 16 17 M Check that Buffer AL and Buffer ATL do not contain a white precipitate If necessary incubate Buffer AL and Buffer ATL for 30 minutes at 37 C with occasional shaking to dissolve precipitate E Ensure that you are familiar with operating the BioSprint 96 Refer to the BioSprint 96 User Manual for operating instructions M 96 rod covers are supplied either as packets of two or as packets of one inserted into an S Block If using a new packet of two store the second 96 rod cover on another plate It is important that the 96 rod cover does not become bent Things to do before starting M Set a shaker incubator with an adapter for S Blocks or 1 5 ml microcentrifuge tubes if lysis is not to be performed in an S Block to 56 C for use in step 3 of the procedure E MagAttract magnetic particles copurify RNA and DNA if both are present in the sample If RNA free DNA is required add RNase A to the sample in st
7. blocks as instructed by the display of the BioSprint 96 Press Start after removing each plate or block The first item to be removed contains the purified samples Carryover of magnetic particles in eluates will not affect most downstream applications If the risk of magnetic particle carryover needs to be minimized the microplate containing eluates should first be placed in a suitable magnet and the eluates transferred to a clean microplate see the appendix page 50 Press Stop after all plates and blocks are removed 12 Discard the used plates blocks and 96 rod cover according to your local safety regulations Note See page 5 for safety information Switch off the BioSprint 96 at the power switch Wipe the worktable and adjacent surfaces using a soft cloth or tissue moistened with distilled water or detergent solution If infectious material is spilt on the worktable clean using 70 ethanol or other disinfectant Note Do not use bleach as disinfectant See page 5 for safety information BioSprint 96 DNA Handbook 06 2012 Protocol Purification of DNA from Cultured Cells This protocol is for purification of total genomic and mitochondrial DNA from up to 5 x 10 diploid cells per sample using the BioSprint 96 workstation and the BioSprint 96 DNA Blood Kit Important points before starting M Tape sheets are required for this protocol See Equipment and Reagents to Be Supplied by User page 9
8. is stable for up to 2 months when stored at 2 8 C Storing reconstituted QIAGEN Protease at room temperature for prolonged periods should be avoided Reconstituted QIAGEN Protease can be stored at 20 C for up to 6 months but repeated freezing and thawing should be avoided We recommend dividing the reconstituted QIAGEN Protease into aliquots before storing at 20 C Intended Use The BioSprint 96 DNA Blood Kit is intended for molecular biology applications This product is 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 safety data sheets SDSs These are available online in convenient and compact PDF format at www qiagen com ts msds asp where you can find view and print the SDS for each QIAGEN kit and kit component CAUTION DO NOT add bleach or acidic solutions directly to the sample preparation waste Buffers AL and AW1 contain guanidine hydrochloride which can form highly reactive compounds when combined with bleach If liquid containing these buffers is spilt clean with s
9. label i e well Al faces inward 12 Check that the protective cover is correctly installed it should fit exactly into the body of the BioSprint 96 Slide the door shut to protect samples from contamination See the BioSprint 96 User Manual for safety information 13 Press Start to start sample processing BioSprint 96 DNA Handbook 06 2012 21 14 After the samples are processed remove the plates and blocks as instructed by the display of the BioSprint 96 Press Start after removing each plate or block The first item to be removed contains the purified samples Carryover of magnetic particles in eluates will not affect most downstream applications If the risk of magnetic particle carryover needs to be minimized the microplate containing eluates should first be placed in a suitable magnet and the eluates transferred to a clean microplate see the appendix page 50 15 Press Stop after all plates and blocks are removed 16 Discard the used plates blocks and 96 rod cover according to your local safety regulations Note See page 5 for safety information 17 Switch off the BioSprint 96 at the power switch 18 Wipe the worktable and adjacent surfaces using a soft cloth or tissue moistened with distilled water or detergent solution If infectious material is spilt on the worktable clean using 70 ethanol or other disinfectant Note Do not use bleach as disinfectant See page 5 for safety information
10. on the BioSprint 96 workstation Press Start to start the protocol run 15 The LCD displays a message asking you to load slot 8 of the worktable with the 96 rod cover see Table 11 page 45 After loading slot 8 press Start The worktable rotates and a new message appears asking you to load slot 7 with the elution plate Load slot 7 and press Start again Continue this process of pressing Start and loading a particular slot until all slots are loaded Note Each slot is labeled with a number Load each 96 well plate or S Block so that well A1 is aligned with the slot s label i e well Al faces inward 16 Check that the protective cover is correctly installed it should fit exactly into the body of the BioSprint 96 Slide the door shut to protect samples from contamination See the BioSprint 96 User Manual for safety information 17 Press Start to start sample processing 46 BioSprint 96 DNA Handbook 06 2012 18 After the samples are processed remove the plates and blocks as instructed by the display of the BioSprint 96 Press Start after removing each plate or block The first item to be removed contains the purified samples Carryover of magnetic particles in eluates will not affect most downstream applications If the risk of magnetic particle carryover needs to be minimized the microplate containing eluates should first be placed in a suitable magnet and the eluates transferred to
11. or at 20 C for longer periods Minimizing magnetic particle carryover in the DNA If the purified DNA is to be analyzed by real time PCR any trace amounts of magnetic particles should be minimized using a magnet Transfer the eluates to 1 5 ml microcentrifuge tubes Apply the tubes to a suitable magnet e g QIAGEN 12 Tube Magnet for 10 minutes and carefully remove the supernatants Alternatively transfer the eluates to a flat bottom microplate e g QIAGEN 96 Well Microplate FB Apply the microplate to a suitable magnet e g QIAGEN 96 Well Magnet Type A for 10 minutes and carefully remove the supernatants If a suitable magnet is not available transfer the eluates to microcentrifuge tubes centrifuge for 1 minute at full speed to pellet any remaining magnetic particles and carefully remove the supernatants Quantification and determination of purity of DNA The concentration of DNA should be determined by measuring the absorbance at 260 nm A250 in a spectrophotometer Absorbance readings at 260 nm should fall between 0 1 and 1 0 to be accurate An absorbance of 1 unit at 260 nm corresponds to 50 ug of DNA per ml A260 1 gt 50 ug ml Use a low salt buffer of neutral pH e g 10 mM Tris HCI pH 7 to dilute DNA samples and to calibrate the spectrophotometer The ratio between the absorbance values at 260 nm and 280 nm gives an estimate of DNA purity For accurate results use a slightly alkaline buffer e g 10 mM Tri
12. procedure The BioSprint 96 DNA Blood Kit uses MagAttract magnetic particle technology for DNA purification MagAttract technology combines the speed and efficiency of silica based DNA purification with the convenient handling of magnetic particles see flowchart next page DNA binds to the silica surface of MagAttract magnetic particles in the presence of a chaotropic salt DNA bound to the magnetic particles is then efficiently washed Two different wash buffers are used followed by a rapid rinse with distilled water or an air drying step which considerably improves the purity of the DNA High quality DNA is eluted in Buffer AE DNA yields depend on sample type sample storage and if purifying from whole blood white blood cell content Supplementary protocols for processing other sample types or for purification of different target molecules using the BioSprint 96 workstation are available at www giagen com literature protocols or from QIAGEN Technical Services BioSprint Software protocols for automated sample processing are available from QIAGEN Technical Services BioSprint 96 DNA Handbook 06 2012 7 BioSprint 96 DNA Procedure Standard protocol Rapid blood protocol Sample Lysis Add isopropanol and MagAttract Suspension G Add sample Transfer to and reagents S Block to S Block DNA binds to magnetic particles Magnetic separation Wash Magnetic separation Elute an lt Gis Pure DNA Rapid
13. should be avoided Briefly centrifuge the S Block or 1 5 ml microcentrifuge tube to remove drops from underneath the tape or lid Remove the tape sheet from the S Block If lysis was performed in microcentrifuge tubes transfer the lysates to an S Block Vortex the master mix containing isopropanol and MagAttract Suspension G for 1 min see Things to do before starting Add 230 pl master mix to each sample in the S Block Note If using a multidispenser add 225 ul master mix to each sample Switch on the BioSprint 96 at the power switch Slide open the front door of the protective cover Select the protocol BS96 DNA Blood 200 using the A and v keys on the BioSprint 96 workstation Press Start to start the protocol run The LCD displays a message asking you to load slot 8 of the worktable with the 96 rod cover see Table 7 page 29 After loading slot 8 press Start The worktable rotates and a new message appears asking you to load slot 7 with the elution plate Load slot 7 and press Start again Continue this process of pressing Start and loading a particular slot until all slots are loaded Note Each slot is labeled with a number Load each plate or block so that well A1 is aligned with the slot s label i e well Al faces inward Check that the protective cover is correctly installed it should fit exactly into the body of the BioSprint 96 Slide the door shut to protect samples f
14. slots 7 and 8 according to Table 6 on the next page The S Blocks and microplates are loaded onto the worktable in step 7 In each plate or block the number of wells to be filled with buffer should match the number of samples to be processed e g if processing 48 samples fill 48 wells per plate or block Ensure that buffers are added to the same positions in each plate or block e g if processing 48 samples fill wells AI H1 to A6 H6 of each plate or block S En nn 24 BioSprint 96 DNA Handbook 06 2012 Table 6 BioSprint 96 worktable setup and reagent volumes Volume per well ul Message when Slot loading Plate block To add 8 Load 96 well Large 96 Rod Cover microplate Rod Cover MP 7 Load Elution 96 well Buffer AE 100 200 microplate MP 6 Load S Block RNase free 500 500 Wash 5 water 5 Load S Block Buffer AW2 500 500 Wash 4 4 Load S Block Buffer AW2 500 500 Wash 3 3 Load S Block Buffer AW1 500 500 Wash 2 2 Load S Block Buffer AW1 500 650 Wash 1 1 Load Lysate S Block Lysatet 325 650 Contains 0 02 v v Tween 20 t Added at steps 2 and 3 includes volume of QIAGEN Protease sample Buffer AL isopropanol and MagAttract Suspension G of an S Block Add W 100 pl or 200 ul sample to the QIAGEN Protease Note Record in which wells you load the samples 3 Vortex the master mix containing Buffer AL isopropanol and MagAttract Suspension G for 1 min see Things to do befo
15. 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 2004 2012 QIAGEN all rights reserved mb _ _ _ _ _ Tm m www giagen com Australia techservice au giagen com Austria techservice at qiagen com Belgium techservice bnl giagen com Brazil suportetecnico brasil giagen com Canada techservice ca giagen com China techservice cn qiagen com Denmark techservice nordic qiagen com Finland techservice nordic giagen com France techservice fr qiagen com Germany techservice de giagen com Hong Kong techservice hk giagen com India techservice india giagen com Ireland techservice uk qiagen com Italy techservice it qiagen com Japan techservice jp qiagen 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 giagen com Switzerland techservice ch qiagen com amp UK techservice uk giagen com USA techservice us qiagen com QIAGE N en Sample amp Assay Technolog
16. 0 ul blood samples choose if processing 200 ul blood samples Things to do before starting E Thaw and equilibrate up to 96 whole blood samples at room temperature 15 25 C BE All samples in a single procedure must have the same volume 100 ul or 200 ul If the volume of a sample needs to be increased add the appropriate volume of PBS E MagAttract magnetic particles copurify RNA and DNA if both are present in the sample If RNA free DNA is required add RNase A to the sample before starting the procedure The final RNase A concentration should be 2 mg ml e g add 4 ul of a 100 mg ml RNase A solution to a 200 ul sample BioSprint 96 DNA Handbook 06 2012 23 M Prepare a master mix according to the table below for use in step 3 of the procedure Before adding MagAttract Suspension G ensure that it is fully resuspended Vortex for 3 minutes before using for the first time and for minute before subsequent uses Note Prepare a volume of master mix 10 greater than that required for the total number of sample purifications to be performed If using a multidispenser W 225 ul or 450 ul master mix is required per sample see step 3 of the procedure The starting volume of master mix should be increased accordingly Volume of reagent per sample wl Reagent a Buffer AL 100 200 lsopropanol 100 200 MagAttract Suspension G 15 30 Procedure 1 Prepare five S Blocks slots 2 6 and two 96 well microplates
17. 00 pl of the lysate from each well to a fresh S Block Do not transfer the swabs to the fresh S Block 7 Vortex the master mix containing Buffer AL isopropanol and MagAttract Suspension G for 1 min see Things to do before starting Add 420 pl master mix to each sample in the S Block Note If using a multidispenser add 400 ul master mix to each sample 8 Switch on the BioSprint 96 at the power switch 9 Slide open the front door of the protective cover 42 BioSprint 96 DNA Handbook 06 2012 10 Select the protocol BS96 DNA Swab using the A and v keys on the BioSprint 96 workstation Press Start to start the protocol run 11 The LCD displays a message asking you to load slot 8 of the worktable with the 96 rod cover see Table 10 page 42 After loading slot 8 press Start The worktable rotates and a new message appears asking you to load slot 7 with the elution plate Load slot 7 and press Start again Continue this process of pressing Start and loading a particular slot until all slots are loaded Note Each slot is labeled with a number Load each 96 well plate or S Block so that well A1 is aligned with the slot s label i e well Al faces inward 12 Check that the protective cover is correctly installed it should fit exactly into the body of the BioSprint 96 Slide the door shut to protect samples from contamination Warning To avoid contact with moving parts during operation of th
18. 6 includes volume of sample QIAGEN Proteinase K Buffer ATL Buffer AL isopropanol and MagAttract Suspension G Briefly centrifuge the S Block or microcentrifuge tube containing the sample to remove drops from underneath the tape or inside the lid Remove the tape sheet from the S Block If lysis was performed in microcentrifuge tubes transfer the lysates to an S Block Vortex the master mix containing Buffer AL isopropanol and a MagAttract Suspension G for 1 min see Things to do before starting Add 430 pl of master mix to each sample in the S Block Note If using a multidispenser add 450 ul master mix to each sample Switch on the BioSprint 96 at the power switch Slide open the front door of the protective cover Select the protocol BS96 DNA Tissue using the A and y keys on the BioSprint 96 workstation Press Start to start the protocol run BioSprint 96 DNA Handbook 06 2012 10 The LCD displays a message asking you to load slot 8 of the worktable with the 96 rod cover see Table 9 page 38 After loading slot 8 press Start The worktable rotates and a new message appears asking you to load slot 7 with the elution plate Load slot 7 and press Start again Continue this process of pressing Start and loading a particular slot until all slots are loaded Note Each slot is labeled with a number Load each plate or block so that well Al is aligned with the slot s label i e
19. A from Buccal Swabs 40 E Purification of DNA from Dried Blood Spots 44 Troubleshooting Guide 48 Appendix Handling Quantification and Determination of Purity of DNA 50 Ordering Information 52 BioSprint 96 DNA Handbook 06 2012 3 Kit Contents Quick Start Protocol When each prep is from 200 ul blood t Contains a guanidine salt Not compatible with disinfectants containing bleach See page 5 for safety information Resuspension volume 1 2 ml Resuspension volume 4 4 ml 1 BioSprint 96 DNA Blood Kit 48 384 Catalog no 940054 940057 Number of preps 48 384 Buffer ALt 12 ml 3 x 33 ml QIAGEN Protease 1 vial 2 vials Protease Solvent 1 2 ml 2x4 4 ml MagAttract Suspension G 1 6 ml 13 ml Buffer AW1 concentrate 27 ml 2 x 98 ml Buffer AW2 concentrate 17 ml 2x 81 ml RNase free water 4x10ml 2 x 250 ml Buffer AE 15 ml 128 ml Large 96 Rod Cover 1 4 96 Well Microplate MP 2 8 S Block 6 24 1 1 Contains sodium azide as a preservative Storage All buffers and reagents can be stored dry at room temperature 15 25 C for up to 1 year without showing any reduction in performance Lyophilized QIAGEN Protease can be stored dry at room temperature for up to 1 year without any decrease in performance For storage longer than 1 year or if ambient temperatures constantly exceed 25 C QIAGEN Protease should be stored dry at 2 8 C 4 BioSprint 96 DNA Handbook 06 2012 Reconstituted QIAGEN Protease
20. June 2012 BioSprint 96 DNA Handbook For purification of DNA from human whole blood animal whole blood buffy coat cultured cells tissues rodent tails buccal swabs dried blood spots using the BioSprint 96 workstation 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 5 Safety Information 5 Quality Control 6 Introduction 7 Principle and procedure 7 Equipment and Reagents to Be Supplied by User 9 Important Notes 11 Starting material 11 Storing blood samples 11 Preparing buffy coat 12 Yield and quality of purified DNA 12 Preparing reagents 16 Quantification of DNA 17 Protocols E Purification of DNA from Blood 18 E Rapid Purification of DNA from Human Whole Blood 23 E Purification of DNA from Cultured Cells 27 E Purification of DNA from Tissues 32 E Purification of DNA from Rodent Tails 36 m Purification of DN
21. Suspension G Buffers and Reagents For 384 preps Large 96 Rod Covers 96 Well Microplates MP S Blocks MagAttract Suspension G Buffers and Reagents 200 ml Tissue Lysis Buffer for 1000 preps 2 ml gt 600 mAU ml solution 10 ml gt 600 mAU mIl solution 240 ml Elution Buffer Adhesive tape sheets for sealing multiwell plates and blocks 25 sheets per pad 5 pads per pack 16 x 96 Rod Covers for use with the BioSprint 96 96 well microplates 20 per case for use with the BioSprint 96 96 well blocks with 2 2 ml wells 24 per case for use with the BioSprint 96 Magnet for separating magnetic particles in 12 x 1 5 ml or 2 ml tubes Magnet for separating magnetic particles in wells of 96 well plates 2 x 96 Well Microplates FB 96 well microplates with flat bottom wells for use with the 96 Well Magnet Type A pack of 24 Cat no 940054 940057 19076 19131 19133 19077 19570 1031668 1031656 19585 36912 36915 36985 BioSprint 96 DNA Handbook 06 2012 Product Contents Centrifuges Centrifuge 4 16 Universal laboratory centrifuge with brushless motor Centrifuge 4 16K Refrigerated universal laboratory centrifuge with brushless motor Plate Rotor 2 x 96 Rotor for 2 QIAGEN 96 well plates for use with QIAGEN Centrifuges Related products BioSprint 15 DNA Blood Kits for rapid purification of DNA from cells tissue blood buffy coat buccal swabs and dried blood spots using th
22. a clean microplate see the appendix page 50 19 Press Stop after all plates and blocks are removed 20 Discard the used plates blocks and 96 rod cover according to your local safety regulations Note See page 5 for safety information 21 Switch off the BioSprint 96 at the power switch 22 Wipe the worktable and adjacent surfaces using a soft cloth or tissue moistened with distilled water or detergent solution If infectious material is spilt on the worktable clean using 70 ethanol or other disinfectant Note Do not use bleach as disinfectant See page 5 for safety information i E BioSprint 96 DNA Handbook 06 2012 47 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 qiagen com Comments and suggestions Low DNA yield a Inefficient cell lysis due Repeat the DNA purification procedure with a to insufficient mixing of new sample Be sure to mix the sample and the sample with Buffer AL immediately and thoroughly by Buffer AL pulse vortexing 2 Inefficient cell lysis due Repeat the DNA
23. active tissues such as liver and kidney contain high levels of RNA which will copurify with genomic DNA If RNA free genomic DNA is required add 4 ul of RNase A 100 mg ml mix by vortexing and incubate for 2 min at room temperature 15 25 C 4 Towards the end of proteinase K digestion prepare five S Blocks slots 2 6 and two 96 well microplates slots 7 and 8 according to Table 8 on the next page The S Blocks and microplates are loaded onto the worktable in step 10 In each plate or block the number of wells to be filled with buffer should match the number of samples to be processed e g if processing 48 samples fill 48 wells per plate or block Ensure that buffers are added to the same positions in each plate or block e g if processing 48 samples fill wells AI H1 to A6 H6 of each plate or block BioSprint 96 DNA Handbook 06 2012 33 Table 8 BioSprint 96 worktable setup and reagent volumes Message Volume when per well Slot loading Plate block To add ul 8 Load 96 well Large 96 rod cover Rod Cover microplate MP 7 Load Elution 96 well Buffer AE 200 microplate MP 6 Load S Block RNase free water 500 Wash 5 5 Load S Block Buffer AW2 500 Wash 4 4 Load S Block Buffer AW2 500 Wash 3 3 Load S Block Buffer AW1 500 Wash 2 2 Load S Block Buffer AW1 650 Wash 1 1 Load Lysate S Block Lysatet 630 Contains 0 02 v v Tween 20 t Added at steps 1 2 and 6 includes volume of sample QIAGEN Pr
24. containing concentrated leukocytes and the bottom layer contains concentrated erythrocytes Approximately 1 ml leukocyte containing fraction should be harvested from 10 ml centrifuged whole blood which gives 10x enrichment To avoid overloading the DNA purification procedure do not prepare buffy coat samples of gt 10x enrichment If buffy coat samples are of gt 10x enrichment use less starting material in the DNA purification procedure Yield and quality of purified DNA DNA yields depend on the sample type the number of nucleated cells in the sample and the protocol used for DNA purification Typical DNA yields obtained from a variety of sample types are shown in Table 2 page 13 Elution in smaller volumes increases the final DNA concentration in the eluate but slightly reduces overall DNA yield We recommend using an elution volume appropriate for the intended downstream application The BioSprint 96 DNA procedure yields pure DNA with A260 A280 ratios greater than 1 7 The purified DNA is up to 50 kb in size Figures 1 and 2 pages 14 and 15 and is suitable for all downstream applications including Southern blotting PCR and real time PCR Figure 3 page 15 12 BioSprint 96 DNA Handbook 06 2012 Table 2 Typical DNA yields from a range of sample types Sample type Amount of sample Typical DNA yield ug Bovine tissue Muscle 25 mg 13 5 1 5 Heart 25 mg 3 4 0 6 Spleen 25 mg 59 1 4 8 Lung 25 mg 14 7 5 5 Liver 25
25. e BioSprint 96 do not insert your hands and fingers inside the workstation See the BioSprint 96 User Manual for safety information 13 Press Start to start sample processing 14 After the samples are processed remove the plates and blocks as instructed by the display of the BioSprint 96 Press Start after removing each plate or block The first item to be removed contains the purified samples Carryover of magnetic particles in eluates will not affect most downstream applications If the risk of magnetic particle carryover needs to be minimized the microplate containing eluates should first be placed in a suitable magnet and the eluates transferred to a clean microplate see the appendix page 50 15 Press Stop after all plates and blocks are removed 16 Discard the used plates blocks and 96 rod cover according to your local safety regulations Note See page 5 for safety information 17 Switch off the BioSprint 96 at the power switch 18 Wipe the worktable and adjacent surfaces using a soft cloth or tissue moistened with distilled water or detergent solution If infectious material is spilt on the worktable clean using 70 ethanol or other disinfectant Note Do not use bleach as disinfectant See page 5 for safety information BioSprint 96 DNA Handbook 06 2012 43 Protocol Purification of DNA from Dried Blood Spots This protocol is for purification of total genomic and mitochondrial DNA from bl
26. e BioSprint 15 workstation BioSprint 15 DNA For 45 preps 5 Rod Covers 5 Tube Blood Kit 45 Strips MagAttract Suspension G Buffers and Reagents BioSprint DNA Plant Kits for rapid purification of total DNA from plant tissue using BioSprint workstations BioSprint 15 DNA Plant For 360 preps 5 Rod Covers 5 Tube Kit 360 Strips MagAttract Suspension G Buffers and Reagents BioSprint 96 DNA Plant For 576 preps Large 96 Rod Covers Kit 576 96 Well Microplates MP S Blocks MagAttract Suspension G Buffers and Reagents Cat no 81300 81310 813254 813208 81400 814108 81425 81420 81031 940014 941517 941557 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 Japan t North America t UK 8 Rest of World Other kit sizes are available see www giagen com BioSprint 96 DNA Handbook 06 2012 53 Notes 54 BioSprint 96 DNA Handbook 06 2012 Trademarks QIAGEN BioSprint MagAttract QuantiTect Rotor Gene QIAGEN Group 903 FTA Whatman Whatman International Ltd DACRON E INVISTA North America S A R L Corporation Eppendorf Eppendorf AG Finnpipette Thermo Electron Oy Corporation Puritan Puritan Medical Products Com
27. e water 500 Wash 5 5 Load S Block Buffer AW2 500 Wash 4 4 Load S Block Buffer AW2 500 Wash 3 3 Load S Block Buffer AW1 500 Wash 2 2 Load S Block Buffer AW1 650 Wash 1 1 Load Lysate S Block Lysatet 640 Contains 0 02 v v Tween 20 t Added at steps 1 2 3 7 10 and 11 includes volume of QIAGEN Proteinase K Buffer ATL Buffer AL isopropanol and MagAttract Suspension G BioSprint 96 DNA Handbook 06 2012 45 6 Briefly centrifuge the S Block containing the samples to remove drops from underneath the tape Remove the tape sheet from the S Block 7 Add 200 pl Buffer AL seal the S Block with a tape sheet and mix by pulse vortexing for 10 s Note Make sure that the punches are fully covered with buffer If necessary briefly centrifuge the S Block after mixing 8 Place the S Block in a shaker incubator and incubate at 56 C with shaking at 900 rpm for 10 min 9 Briefly centrifuge the S Block containing the samples to remove drops from underneath the tape Remove the tape sheet from the S Block 10 Add 200 ul isopropanol 11 Add 20 pl MagAttract Suspension G Before adding MagAttract Suspension G ensure that it is fully resuspended Vortex for 3 min before using for the first time and for 1 min before subsequent uses 12 Switch on the BioSprint 96 at the power switch 13 Slide open the front door of the protective cover 14 Select the protocol BS96 DNA Dried Blood using the A and v keys
28. ed Buffer AW2 at room temperature 15 25 C Note Always mix reconstituted Buffer AW2 before use by shaking the bottle 5 times MagAttract Suspension G To ensure that the magnetic silica particles are fully resuspended MagAttract Suspension G must be shaken and vortexed before use Before the first use shake the vial or bottle and vortex for 3 minutes Before subsequent uses shake the bottle and vortex for 1 minute RNase free water In the BioSprint 96 DNA procedure magnetic particles are briefly washed with RNase free water to remove residual ethanol from the previous wash step with Buffer AW2 Tween 20 must be added to the RNase free water to a final concentration of 0 02 v v e g add 6 ul and 50 ul Tween 20 to 30 ml and 250 ml RNase free water respectively Quantification of DNA Carryover of magnetic particles may affect the absorbance reading at 260 nm A260 of the purified DNA but should not affect downstream applications The measured absorbance at 320 nm A329 should be subtracted from all absorbance readings See the appendix page 50 for more information BioSprint 96 DNA Handbook 06 2012 17 Protocol Purification of DNA from Blood This protocol is for purification of total genomic and mitochondrial DNA from whole blood or blood products using the BioSprint 96 workstation and the BioSprint 96 DNA Blood Kit Human blood samples can be 100 ul 200 ul or 250 ul Animal blood samples containing non nucleat
29. ed erythrocytes can be 100 ul or 200 ul Buffy coat samples can be 100 ul or 200 ul Important points before starting E Tape sheets are required for this protocol See Equipment and Reagents to Be Supplied by User page 9 M Check that QIAGEN Protease Buffer AW1 Buffer AW2 and RNase free water have been prepared according to the instructions on pages 16 17 M Check that Buffer AL does not contain a white precipitate by shaking the bottle If necessary incubate for 30 minutes at 37 C with occasional shaking to dissolve precipitate M Blood samples must be in the range of 100 250 ul Animal blood samples must be in the range 100 200 ul If necessary the volume of animal blood used can be reduced and the sample volume adjusted to 100 ul or 200 ul with Buffer AE Since bird and fish blood contain nucleated erythrocytes use less than 20 ul blood and adjust the sample volume to 200 ul with Buffer AE Buffy coat samples must be 100 200 ul E Ensure that you are familiar with operating the BioSprint 96 Refer to the BioSprint 96 User Manual for operating instructions M 96 rod covers are supplied either as packets of two or as packets of one inserted into an S Block If using a new packet of two store the second 96 rod cover on another plate It is important that the 96 rod cover does not become bent E In some steps of the procedure one of three choices can be made Choose W if processing 100 ul blood samples choose if process
30. ep 3 of the procedure The final RNase A concentration should be 2 mg ml e g add 4 ul of a 100 mg ml RNase A solution to a 200 ul sample E Prepare a master mix according to the table on the next page for use in step 6 of the procedure Before adding MagAttract Suspension G ensure that it is fully resuspended Vortex for 3 minutes before using for the first time and for 1 minute before subsequent uses Note Prepare a volume of master mix 10 greater than that required for the total number of sample purifications to be performed If using a multidispenser 450 ul master mix is required per sample see step 6 of the procedure The starting volume of master mix should be increased accordingly een 36 BioSprint 96 DNA Handbook 06 2012 Reagent Volume of reagent per sample ul Buffer AL 200 lsopropanol 200 MagAttract Suspension G 30 Procedure 1 Cut lt 1 2 cm approximately 25 mg of each rodent tail sample into small pieces Place the tissue sample into the well of an S Block or into a 1 5 ml microcentrifuge tube not supplied and add 180 ul Buffer ATL not supplied 2 Add 20 pl QIAGEN Proteinase K not supplied and seal the S Block with a tape sheet not supplied or close the microcentrifuge tube 3 Place the S Block or 1 5 ml microcentrifuge tube in a shaker incubator and incubate at 56 C with shaking until the tissue is completely lysed Lysis time varies depending on the type of tissue processed L
31. epare a master mix according to the table on the next page for use in step 6 of the procedure Before adding MagAttract Suspension G ensure that it is fully resuspended Vortex for 3 minutes before using for the first time and for 1 minute before subsequent uses Note Prepare a volume of master mix 10 greater than that required for the total number of sample purifications to be performed If using a multidispenser 450 ul master mix is required per sample see step 6 of the procedure The starting volume of master mix should be increased accordingly S En nn 32 BioSprint 96 DNA Handbook 06 2012 Reagent Volume of reagent per sample ul Buffer AL 200 lsopropanol 200 MagAttract Suspension G 30 Procedure 1 Cut lt 25 mg of each tissue sample into small pieces Place the tissue sample into the well of an S Block or into a 1 5 ml microcentrifuge tube not supplied and add 180 pl Buffer ATL not supplied 2 Add 20 pl QIAGEN Proteinase K not supplied and seal the S Block with a tape sheet not supplied or close the 1 5 ml microcentrifuge tube 3 Place the S Block or 1 5 ml microcentrifuge tube in a shaker incubator and incubate at 56 C with shaking until the tissue is completely lysed Lysis time varies depending on the type of tissue processed Lysis is usually complete in 1 3 h If it is more convenient samples can be lysed overnight this will not affect the DNA quality Optional Transcriptionally
32. ies
33. ing 200 ul blood samples choose if processing 250 ul blood samples Things to do before starting E Thaw and equilibrate up to 96 whole blood samples at room temperature 15 25 C or prepare buffy coat samples according to page 12 M Set a shaker incubator with an adapter for S Blocks to 70 C for use in step 4 of the procedure 18 BioSprint 96 DNA Handbook 06 2012 E All samples in a single procedure must have the same volume 100 ul 200 ul or 250 ul If the volume of a sample needs to be increased add the appropriate volume of PBS human blood samples or Buffer AE animal bird and fish blood samples E MagAttract magnetic particles copurify RNA and DNA if both are present in the sample If RNA free DNA is required add RNase A to the sample before starting the procedure The final RNase A concentration should be 2 mg ml e g add 4 ul of a 100 mg ml RNase A solution to a 200 ul sample Procedure 1 Prepare five S Blocks and two 96 well microplates according to Table 5 on the next page The S Blocks and microplates are loaded onto the worktable in step 11 In each plate or block the number of wells to be filled with buffer should match the number of samples to be processed e g if processing 48 samples fill 48 wells per plate or block Ensure that buffers are added to the same positions in each plate or block e g if processing 48 samples fill wells AI H1 to A6 H6 of each plate or block BioSprint 96 DNA
34. ion Note Do not add QIAGEN Protease directly to Buffer AL 4 Incubate at 70 C for 10 min Maximum DNA yields are achieved after lysis at 70 C for 10 min Longer incubation times should be avoided 5 Briefly centrifuge the S Block to remove drops from underneath the tape Remove the tape sheet from the S Block 6 Add 100 ul 200 ul or 250 pl isopropanol 7 Add 15 ul 30 ul or 35 ul MagAttract Suspension G Note Before adding MagAttract Suspension G ensure that it is fully resuspended Vortex for 3 min before using for the first time and for 1 min before subsequent uses 8 Switch on the BioSprint 96 at the power switch 9 Slide open the front door of the protective cover 10 Select the protocol W BS96 DNA Blood 100 BS96 DNA Blood 200 or BS96 DNA Blood 250 using the A and v keys Press Start to start the protocol run 11 The LCD displays a message asking you to load slot 8 of the worktable with the 96 rod cover After loading slot 8 press Start The worktable rotates and a new message appears asking you to load slot 7 with the elution plate Load slot 7 and press Start again Continue this process of pressing Start and loading a particular slot until all slots are loaded Table 5 page 20 shows in which slots the plates and blocks should be loaded Note Each slot is labeled with a number Load each plate or block so that well Al is aligned with the slot s
35. ired if processing swabs or dried blood spots E Swabs such as sterile Omni Swabs available from Whatman www whatman com or Puritan applicators with plastic shafts and cotton or Dacron tips available from Hardwood Products www hwppuritan com if processing buccal swabs This is not a complete list of suppliers and does not include many important vendors of biological supplies t Do not use denatured alcohol which contains other substances such as methanol or methylethylketone BioSprint 96 DNA Handbook 06 2012 9 M Filter paper e g 903 Specimen Collection Paper Blood Stain Card or FTA Card Whatman www whatman com or comparable blood cards if processing dried blood spots We recommend using 903 Specimen Collection Paper with the BioSprint 96 workstation BE Shaker incubator e g Eppendorf Thermomixer Comfort cat no 5355 000 011 with adapter for deep well blocks S Blocks cat no 5363 000 012 not needed for rapid blood protocol E Additional S Blocks 24 cat no 19585 if processing swabs E Multichannel pipettor and disposable pipet tips with aerosol barriers 200 ul e g Finnpipette Digital and Finntip Filters see www thermo com if processing swabs E Centrifuge with rotor capable of holding S Blocks e g Centrifuge 4 16 cat nos 81300 81310 81325 and 81320 and Plate Rotor 2 x 96 cat no 81031 from QIAGEN E Manual paper punch 6 mm 1 4 inch if processing dried blood sp
36. mg 74 0 22 3 Kidney 25 mg 33 5 5 4 Mouse tissue Tail 1 2 cm 25 mg 30 9 45 Cultured cells HL 60 2 x 10 cells 9 6 5 6 Blood Human 200 ul 4 5 9 0 5 7 x 10 cells ml Horse 200 ul 4 0 4 9 Bovine 200 ul 4 4 7 0 Sheep 200 ul 1 2 8 0 Pig 200 ul 4 5 9 0 Dog 200 ul 6 9 16 Cat 100 ul 2 9 6 3 Mouse 100 ul 1 5 3 0 Rat 100 ul 1 0 3 0 Birdt 10 ul 147229 Genomic DNA was purified from the indicated samples t Sample volume adjusted to 200 ul with Buffer AE Table 2 continued on next page BioSprint 96 DNA Handbook 06 2012 13 Table 2 continued from previous page Sample type Amount of sample Typical DNA yield ug Dried blood spots 903 Specimen 1 punch 6 mm 1 4 inch 0 10 Collection Paper diameter FTA Card 1 punch 6 mm 1 4 inch 0 05 diameter Swabs Buccal swabs 1 swab 0 8 2 0 Genomic DNA was purified from the indicated samples ee Ree Va hm D y a d hg ha pay Ag ha biaia aan aiy bai an Via od u M Fresh M Frozen M Figure 1 Purification of high quality DNA from fresh and frozen blood Human blood was collected and treated with one of 3 anticoagulants heparin H citrate C or EDTA E DNA was purified from 200 ul blood immediately after blood collection Fresh and after one cycle of freezing and thawing Frozen using the BioSprint 96 DNA Blood Kit DNA was eluted in 200 ul elution buffer Eluates 15 ul were run on a 0 8 agarose gel in 1x TBE
37. n be used and may be either fresh or frozen Frozen samples should be thawed quickly in a 37 C water bath with mild agitation to ensure thorough mixing and then equilibrated to room temperature 15 25 C before beginning the procedure Yield and quality of the purified DNA depend on the storage conditions of the blood Fresher blood samples may yield better results For short term storage of up to 10 days collect blood in tubes containing EDTA as an anticoagulant and store at 2 8 C However for applications requiring maximum fragment size such as Southern blotting we recommend storage at 2 8 C for up to 3 days only as low levels of DNA degradation will occur after this time For long term storage over 10 days collect blood in tubes containing a standard anticoagulant preferably EDTA if high molecular weight DNA is required and store at 0 C BioSprint 96 DNA Handbook 06 2012 11 Preparing buffy coat Buffy coat is a leukocyte enriched fraction of whole blood The efficiency of leukocyte enrichment depends on the procedure used to prepare buffy coat and on the accuracy with which the buffy coat layer is extracted Prepare buffy coat by centrifuging whole blood samples containing a standard anticoagulant EDTA citrate or heparin at 900 1100 x g for 10 minutes at room temperature 15 25 C After centrifugation 3 different fractions are distinguishable the upper clear layer is plasma the intermediate layer is buffy coat
38. ng a tape sheet not supplied After incubation briefly centrifuge the S Block to remove drops from underneath the tape 5 Towards the end of proteinase K digestion prepare five S Blocks slots 2 6 and two 96 well microplates slots 7 and 8 according to Table 10 on the next page The S Blocks and microplates are loaded onto the worktable in step 11 In each plate or block the number of wells to be filled with buffer should match the number of samples to be processed e g if processing 48 samples fill 48 wells per plate or block Ensure that buffers are added to the same positions in each plate or block e g if processing 48 samples fill wells AI H1 to A6 H6 of each plate or block BioSprint 96 DNA Handbook 06 2012 41 Table 10 BioSprint 96 worktable setup and reagent volumes Message Volume when per well Slot loading Plate block To add ul 8 Load 96 well Large 96 rod cover Rod Cover microplate MP 7 Load Elution 96 well Buffer AE 200 microplate MP 6 Load S Block RNase free water 500 Wash 5 5 Load S Block Buffer AW2 500 Wash 4 4 Load S Block Buffer AW2 500 Wash 3 3 Load S Block Buffer AW1 500 Wash 2 2 Load S Block Buffer AW1 650 Wash 1 1 Load Lysate S Block Lysatet 620 Contains 0 02 v v Tween 20 t Added at steps 6 and 7 includes volume of QIAGEN Proteinase K Buffer ATL Buffer AL isopropanol and MagAttract Suspension G 6 Using a multichannel pipettor carefully transfer 2
39. ood card punches using the BioSprint 96 workstation and the BioSprint 96 DNA Blood Kit This protocol is suitable for untreated blood or blood treated with anticoagulants such as EDTA citrate or heparin The blood must be spotted and dried on filter paper according to the manufacturer s instructions We recommend using 903 Specimen Collection Paper with the BioSprint 96 Important points before starting E Buffer ATL QIAGEN Proteinase K and tape sheets are required for this protocol See Equipment and Reagents to Be Supplied by User page 9 EB Check that Buffer AW1 Buffer AW2 and RNase free water have been prepared according to the instructions on pages 16 17 M Check that Buffer AL and Buffer ATL do not contain a white precipitate If necessary incubate Buffer AL and Buffer ATL for 30 minutes at 37 C with occasional shaking to dissolve precipitate E Ensure that you are familiar with operating the BioSprint 96 Refer to the BioSprint 96 User Manual for operating instructions M 96 rod covers are supplied either as packets of two or as packets of one inserted into an S Block If using a new packet of two store the second 96 rod cover on another plate It is important that the 96 rod cover does not become bent Things to do before starting M Set a shaker incubator with an adapter for S Blocks to 56 C for use in steps 4 and 8 of the procedure Procedure 1 Cut 6 mm 1 4 inch diameter punches from a dried blood spot
40. oteinase K Buffer ATL Buffer AL isopropanol and MagAttract Suspension G Briefly centrifuge the S Block or 1 5 ml microcentrifuge tube containing the sample to remove drops from underneath the tape or inside the lid Remove the tape sheet from the S Block If lysis was performed in 1 5 ml microcentrifuge tubes transfer the lysates to an S Block Vortex the master mix containing Buffer AL isopropanol and ae MagAttract Suspension G for 1 min see Things to do before starting Add 430 pl of master mix to each sample in the S Block Note If using a multidispenser add 450 ul master mix to each sample Switch on the BioSprint 96 at the power switch Slide open the front door of the protective cover Select the protocol BS96 DNA Tissue using the A and y keys on the BioSprint 96 workstation Press Start to start the protocol run BioSprint 96 DNA Handbook 06 2012 10 The LCD displays a message asking you to load slot 8 of the worktable with the 96 rod cover see Table 8 page 34 After loading slot 8 press Start The worktable rotates and a new message appears asking you to load slot 7 with the elution plate Load slot 7 and press Start again Continue this process of pressing Start and loading a particular slot until all slots are loaded Note Each slot is labeled with a number Load each plate or block so that well Al is aligned with the slot s label i e well Al faces inward
41. ots Optional If lysis steps are not carried out in an S Block EB Shaker incubator e g Eppendorf Thermomixer Comfort cat no 5355 000 011 not needed for the rapid blood protocol E Microcentrifuge tubes 1 5 ml if processing blood not needed for rapid blood protocol cells tissues or rodent tails M Microcentrifuge tubes 2 ml if processing buffy coat M Microcentrifuge This is not a complete list of suppliers and does not include many important vendors of biological supplies 10 BioSprint 96 DNA Handbook 06 2012 Important Notes Starting material The amounts of starting material for use in BioSprint 96 DNA procedures are shown in Table 1 Table 1 Sample volumes used in BioSprint 96 DNA procedures Sample Amount of sample Whole blood 100 250 ul Buffy coat 100 200 ul Cultured cells Up to 5 x 10 diploid cells Tissues Up to 25 mg Rodent tails 1 2 cm approximately 25 mg Dried blood spots 1 2 punches 6 mm 1 4 inch diameter Buccal swabs 1 swab We recommend using 100 200 ul animal blood containing non nucleated erythrocytes If necessary the volume of animal blood used can be reduced and the sample volume adjusted to 200 pl with Buffer AE For blood samples containing nucleated erythrocytes e g samples from bird and fish use less than 20 ul blood and adjust the sample volume to 200 ul with Buffer AE Storing blood samples Whole blood samples treated with EDTA ACD or heparin ca
42. pany Tween ICI Americas Inc Limited License Agreement for BioSprint 96 DNA Blood Kit 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 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 ED 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
43. protocol only available for blood BioSprint 96 DNA Handbook 06 2012 uoypindaid ajduins jpnupw uoyp gt ylund yya woua paypwojnp Ajjny 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 safety data sheets SDSs available from the product supplier E BioSprint 96 workstation cat no 9000852 M Magnetic head for use with large 96 rod covers supplied with the BioSprint 96 E Pipettors and disposable pipet tips with aerosol barriers 20 1000 ul E Multidispenser e g Finnpipette Stepper from Thermo Electron see www thermo com E Ethanol 96 100 E isopropanol 100 E Phosphate buffered saline PBS may be required for diluting samples E Buffer AE cat no 19077 may be required for diluting samples E Tween 20 M Disposable gloves E Vortexer M Soft cloth or tissue and 70 ethanol or other disinfectant to clean the BioSprint worktable E Buffer ATL cat no 19076 if processing tissues rodent tails swabs and dried blood spots M QIAGEN Proteinase K 2 ml cat no 19131 or QIAGEN Proteinase K 10 ml cat no 19133 if processing tissues rodent tails swabs and dried blood spots M Tape Pads 5 cat no 19570 for sealing S Blocks not needed for rapid blood protocol EB DNase free RNase A required if purified DNA needs to be RNA free not requ
44. purification procedure with a to decreased protease new sample and with freshly reconstituted activity QIAGEN Protease Be sure to store QIAGEN Protease at 2 8 C immediately after use Ensure that QIAGEN Protease is not added directly to Buffer AL c No isopropanol added Repeat the DNA purification procedure with a to the lysate before new sample adding MagAttract Suspension G d MagAttract Suspension Before starting the procedure ensure that the G was not completely MagAttract Suspension G is fully resuspended resuspended Vortex for at least 3 min before the first use and for 1 min before subsequent uses e Buffer AW1 or AW2 Ensure that Buffer AW1 and AW2 concentrates prepared incorrectly were diluted with the correct volumes of ethanol 96 100 see pages 16 17 Repeat the DNA purification procedure with a new sample f Frozen blood samples Thaw frozen blood samples quickly in a 37 C were not mixed water bath with mild agitation to ensure properly after thawing thorough mixing 48 BioSprint 96 DNA Handbook 06 2012 Comments and suggestions DNA does not perform well in downstream applications a Insufficient DNA used Quantify the purified DNA by spectrophotometric in downstream measurement of the absorbance at 260 nm see application the appendix page 50 b Excess DNA used in Excess DNA can inhibit some enzymatic downstream reactions Quantify the purified DNA by application spectrophotometric measurement of the ab
45. r AL isopropanol and MagAttract Suspension G 2 Centrifuge the appropriate number of cells up to 5 x 10 per sample for 5 min at 300 x g Discard the supernatant and resuspend the cell pellet in 200 ul PBS not supplied Lysis can either be performed in an S Block or a 1 5 ml microcentrifuge tube not supplied Cells can be grown in an S Block in 1 ml of growth medium and incubated under appropriate growth conditions When using a frozen cell pellet allow cells to thaw until the pellet can be dislodged before adding PBS Ensure that an appropriate number of cells are used in the procedure For cell lines with a high degree of ploidy e g HeLa cells it is recommended to use less than the maximum number of cells Optional RNase treatment of the sample Add 4 ul of RNase A 100 mg ml mix by vortexing and incubate for 2 min at room temperature 15 25 C BioSprint 96 DNA Handbook 06 2012 29 11 12 13 30 Add 20 pl QIAGEN Protease to the sample Add 200 ul Buffer AL and seal the S Block with a tape sheet not supplied or close the 1 5 ml microcentrifuge tube Mix by pulse vortexing for 15 s To ensure efficient lysis it is essential that the sample and Buffer AL are mixed thoroughly to yield a homogenous solution Note Do not add QIAGEN Protease directly to Buffer AL Incubate at 70 C for 10 min Maximum DNA yields are achieved after lysis at 70 C for 10 min Longer incubation times
46. re starting Add W 215 ul or 430 pl master mix to each sample in the S Block Note If using a multidispenser add W 225 ul or 450 ul master mix to each sample BioSprint 96 DNA Handbook 06 2012 Pipet W 10 ul or 20 ul QIAGEN Protease into the bottom of a well 25 vy 9 10 11 13 14 26 Switch on the BioSprint 96 at the power switch Slide open the front door of the protective cover Select the protocol W BS96 DNA Blood 100 or BS96 DNA Blood 200 using the A and v keys Press Start to start the protocol run The LCD displays a message asking you to load slot 8 of the worktable with the 96 rod cover see Table 6 page 25 After loading slot 8 press Start The worktable rotates and a new message appears asking you to load slot 7 with the elution plate Load slot 7 and press Start again Continue this process of pressing Start and loading a particular slot until all slots are loaded Note Each slot is labeled with a number Load each plate or block so that well Al is aligned with the slot s label i e well Al faces inward Check that the protective cover is correctly installed it should fit exactly into the body of the BioSprint 96 Slide the door shut to protect samples from contamination See the BioSprint 96 User Manual for safety information Press Start to start sample processing After the samples are processed remove the plates and
47. re a volume of master mix 10 greater than that required for the total number of sample purifications to be performed BioSprint 96 DNA Handbook 06 2012 27 Reagent Volume of reagent per sample ul lsopropanol 200 MagAttract Suspension G 30 Procedure 1 Prepare five S Blocks slots 2 6 and two 96 well microplates slots 7 and 8 according to Table 7 on the next page The S Blocks and microplates are loaded onto the worktable in step 11 In each plate or block the number of wells to be filled with buffer should match the number of samples to be processed e g if processing 48 samples fill 48 wells per plate or block Ensure that buffers are added to the same positions in each plate or block e g if processing 48 samples fill wells AI H1 to A6 H6 of each plate or block a ae 28 BioSprint 96 DNA Handbook 06 2012 Table 7 BioSprint 96 worktable setup and reagent volumes Message Volume when per well Slot loading Plate block To add ul 8 Load 96 well Large 96 rod cover Rod Cover microplate MP 7 Load Elution 96 well Buffer AE 200 microplate MP 6 Load S Block RNase free water 500 Wash 5 5 Load S Block Buffer AW2 500 Wash 4 4 Load S Block Buffer AW2 500 Wash 3 3 Load S Block Buffer AW1 500 Wash 2 2 Load S Block Buffer AW1 650 Wash 1 1 Load Lysate S Block Lysatet 650 Contains 0 02 v v Tween 20 t Added at steps 2 4 and 7 includes volume of sample QIAGEN Protease Buffe
48. rification of DNA from Buccal Swabs This protocol is for purification of total genomic and mitochondrial DNA from buccal swabs using the BioSprint 96 workstation and the BioSprint 96 DNA Blood Kit The procedure is optimized for air dried buccal swabs with cotton or Dacron tips and brushes or swabs with an ejectable head e g Whatman Omni Swab Other swab types may also be used Important points before starting E Buffer ATL and QIAGEN Proteinase K are required for this protocol If lysis is to be performed overnight tape sheets for sealing the S Block are also required See Equipment and Reagents to Be Supplied by User page 9 HB Check that Buffer AW1 Buffer AW2 and RNase free water have been prepared according to the instructions on pages 16 17 BE Check that Buffer AL and Buffer ATL do not contain a white precipitate If necessary incubate Buffer AL and Buffer ATL for 30 minutes at 37 C with occasional shaking to dissolve precipitate E Ensure that you are familiar with operating the BioSprint 96 Refer to the BioSprint 96 User Manual for operating instructions M 96 rod covers are supplied either as packets of two or as packets of one inserted into an S Block If using a new packet of two store the second 96 rod cover on another plate It is important that the 96 rod cover does not become bent Things to do before starting M Set a shaker incubator with an adapter for S Blocks to 56 C for use in step 4 of the p
49. rocedure E Prepare a master mix according to the table on the next page for use in step 7 of the procedure Before adding MagAttract Suspension G ensure that it is fully resuspended Vortex for 3 minutes before using for the first time and for 1 minute before subsequent uses Note Prepare a volume of master mix 10 greater than that required for the total number of sample purifications to be performed Reagent Volume of reagent per sample ul Buffer AL 200 lsopropanol 200 MagAttract Suspension G 20 40 BioSprint 96 DNA Handbook 06 2012 Procedure 1 Place the swab into the well of an S Block If using an Omni Swab eject the swab head by pressing the end of the inner shaft towards the swab head If using a cotton or Dacron swab separate the swab head from its shaft by hand or by using scissors 2 If using an Omni Swab add 500 pl Buffer ATL not supplied to the well containing the swab If using a cotton or Dacron swab add 400 ul Buffer ATL to the well containing the swab 3 Add 20 ul QIAGEN Proteinase K not supplied and mix by pulse vortexing for 10 s Note Since the S Block is not covered avoid vortexing at high speeds 4 Place the S Block in a shaker incubator and incubate at 56 C with shaking at 900 rpm for 1 h If it is more convenient samples can be lysed overnight this will not affect the DNA quality Note When lysis is performed overnight we recommend sealing the S Block usi
50. rom contamination See the BioSprint 96 User Manual for safety information Press Start to start sample processing BioSprint 96 DNA Handbook 06 2012 14 After the samples are processed remove the plates and blocks as instructed by the display of the BioSprint 96 Press Start after removing each plate or block The first item to be removed contains the purified samples Carryover of magnetic particles in eluates will not affect most downstream applications If the risk of magnetic particle carryover needs to be minimized the microplate containing eluates should first be placed in a suitable magnet and the eluates transferred to a clean microplate see the appendix page 50 15 Press Stop after all plates and blocks are removed 16 Discard the used plates blocks and 96 rod cover according to your local safety regulations Note See page 5 for safety information 17 Switch off the BioSprint 96 at the power switch 18 Wipe the worktable and adjacent surfaces using a soft cloth or tissue moistened with distilled water or detergent solution If infectious material is spilt on the worktable clean using 70 ethanol or other disinfectant Note Do not use bleach as disinfectant See page 5 for safety information U E ae BioSprint 96 DNA Handbook 06 2012 31 Protocol Purification of DNA from Tissues This protocol is for the purification of total genomic and mitochondrial DNA from up to 25 mg of tiss
51. room temperature 15 25 C for prolonged periods should be avoided Reconstituted QIAGEN Protease can be stored at 20 C for up to 6 months but repeated freezing and thawing should be avoided We recommend dividing the reconstituted QIAGEN Protease into aliquots before storing at 20 C Buffer AL Mix Buffer AL thoroughly by shaking before use Buffer AL is stable for up to 1 year when stored at room temperature 15 25 C Note Do not add QIAGEN Protease directly to Buffer AL Buffer AW1 Buffer AW1 is supplied as a concentrate Before using for the first time add ethanol 96 100 as described on the bottle label see also Table 3 Table 3 Preparing Buffer AW1 Volume of AW1 Volume of ethanol to add Final volume concentrate ml ml ml 27 35 62 98 130 228 Tick the check box on the bottle to indicate that ethanol has been added Store reconstituted Buffer AW1 at room temperature 15 25 C Note Always mix reconstituted Buffer AW1 before use by shaking the bottle 5 times 16 BioSprint 96 DNA Handbook 06 2012 Buffer AW2 Buffer AW2 is supplied as a concentrate Before using for the first time add ethanol 96 100 as described on the bottle label see also Table 4 Table 4 Preparing Buffer AW2 Volume of AW2 Volume of ethanol to add Final volume concentrate ml ml ml 17 AO 57 81 190 271 Tick the check box on the bottle to indicate that ethanol has been added Store reconstitut
52. s HCl pH 7 5 to dilute DNA samples and to calibrate the spectrophotometer Pure DNA has an Aj60 Agg9 ratio of 1 7 1 9 When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate safety data sheets SDSs available from the product supplier 50 BioSprint 96 DNA Handbook 06 2012 Carryover of magnetic particles in the eluates may affect the A260 and Aygo readings but should not affect the performance of the DNA in downstream applications Measure the absorbance at 320 nm 280 nm and 260 nm Subtract the absorbance reading obtained at 320 nm from the readings obtained at 260 nm and 280 nm to correct for the presence of magnetic particles Concentration of DNA sample 50 ug ml x A250 A320 x dilution factor Total amount of DNA isolated concentration x volume of sample in ml Pu rity of DNA sample A260 A320 A280 A320 BioSprint 96 DNA Handbook 06 2012 51 Ordering Information Product BioSprint 96 DNA Blood Kit 48 BioSprint 96 DNA Blood Kit 384 Buffer ATL 200 ml QIAGEN Proteinase K 2 ml QIAGEN Proteinase K 10 ml Buffer AE 240 ml Tape Pads 5 Accessories Large 96 Rod Cover 16 96 Well Microplates MP 20 S Blocks 24 12 Tube Magnet 96 Well Magnet Type A 96 Well Microplates FB 24 52 Contents For 48 preps Large 96 Rod Covers 96 Well Microplates MP S Blocks MagAttract
53. sorbance at 260 nm see the appendix page 50 A260 A230 ratio for purified DNA is low a Inefficient cell lysis due Repeat the DNA purification procedure with a to insufficient mixing of new sample Be sure to mix the sample and the sample with Buffer AL immediately and thoroughly by Buffer AL pulse vortexing b Inefficient cell lysis due Repeat the DNA purification procedure with a to decreased protease new sample and with freshly reconstituted activity QIAGEN Protease Be sure to store QIAGEN Protease at 2 8 C immediately after use Ensure that QIAGEN Protease is not added directly to Buffer AL c No isopropanol added Repeat the DNA purification procedure with a to the lysate before new sample adding MagAttract Suspension G d Buffer AW1 or AW2 Ensure that Buffer AW1 and AW2 concentrates prepared incorrectly were diluted with the correct volumes of ethanol 96 100 see pages 16 17 Repeat the DNA purification procedure with a new sample e Absorbance reading at To correct for the presence of magnetic particles 320 nm was not in the eluate an absorbance reading at 320 nm subtracted from the should be taken and subtracted from the absorbance readings at absorbance readings obtained at 260 nm and 260 nm and 280 nm 280 nm see the appendix page 50 BioSprint 96 DNA Handbook 06 2012 49 Appendix Handling Quantification and Determination of Purity of DNA Storage of DNA Purified DNA may be stored at 2 8 C for 24 hours
54. ue per sample using the BioSprint 96 workstation and the BioSprint 96 DNA Blood Kit Important points before starting E Buffer ATL QIAGEN Proteinase K and tape sheets are required for this protocol See Equipment and Reagents to Be Supplied by User page 9 EB Check that Buffer AW1 Buffer AW2 and RNase free water have been prepared according to the instructions on pages 16 17 M Check that Buffer AL and Buffer ATL do not contain a white precipitate If necessary incubate Buffer AL and Buffer ATL for 30 minutes at 37 C with occasional shaking to dissolve precipitate E Ensure that you are familiar with operating the BioSprint 96 Refer to the BioSprint 96 User Manual for operating instructions M 96 rod covers are supplied either as packets of two or as packets of one inserted into an S Block If using a new packet of two store the second 96 rod cover on another plate It is important that the 96 rod cover does not become bent Things to do before starting M Set a shaker incubator with an adapter for S Blocks or 1 5 ml microcentrifuge tubes if lysis is not to be performed in an S Block to 56 C for use in step 3 of the protocol M MagAttract magnetic particles copurify RNA and DNA if both are present in the sample If RNA free DNA is required add RNase A to the sample in step 3 of the procedure The final RNase A concentration should be 2 mg ml e g add 4 ul of a 100 mg ml RNase A solution to a 200 ul sample E Pr
55. uitable 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 If liquid containing potentially infectious agents is spilt on the BioSprint 96 workstation clean the affected area first with laboratory detergent and water and then with 1 v v sodium hypochlorite followed by water 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 BioSprint 96 DNA Handbook 06 2012 5 Quality Control In accordance with QIAGEN s ISO certified Quality Management System each lot of BioSprint 96 DNA Blood Kit is tested against predetermined specifications to ensure consistent product quality 6 BioSprint 96 DNA Handbook 06 2012 Introduction The BioSprint 96 DNA Blood Kit is designed for purification of total DNA i e genomic and mitochondrial DNA from whole blood buffy coat cultured cells tissues rodent tails buccal swabs dried blood spots and other sample types using the BioSprint 96 workstation The BioSprint 96 DNA Blood Kit provides high quality DNA that is free of protein nucleases and other contaminants or inhibitors The DNA is suitable for direct use in downstream applications such as amplification or other enzymatic reactions Principle and
56. well Al faces inward 11 Check that the protective cover is correctly installed it should fit exactly into the body of the BioSprint 96 Slide the door shut to protect samples from contamination See the BioSprint 96 User Manual for safety information 12 Press Start to start sample processing 13 After the samples are processed remove the plates and blocks as instructed by the display of the BioSprint 96 Press Start after removing each plate or block The first item to be removed contains the purified samples Carryover of magnetic particles in eluates will not affect most downstream applications If the risk of magnetic particle carryover needs to be minimized the microplate containing eluates should first be placed in a suitable magnet and the eluates transferred to a clean microplate see the appendix page 50 14 Press Stop after all plates and blocks are removed 15 Discard the used plates blocks and 96 rod cover according to your local safety regulations Note See page 5 for safety information 16 Switch off the BioSprint 96 at the power switch 17 Wipe the worktable and adjacent surfaces using a soft cloth or tissue moistened with distilled water or detergent solution If infectious material is spilt on the worktable clean using 70 ethanol or other disinfectant Note Do not use bleach as disinfectant See page 5 for safety information BioSprint 96 DNA Handbook 06 2012 39 Protocol Pu
57. with a single hole paper punch Place 1 or 2 blood card punches into the well of an S Block Note We do not recommend using punches with a diameter of less than 6 mm 2 Add 200 pl Buffer ATL not supplied 3 Add 20 ul QIAGEN Proteinase K not supplied and seal the S Block with a tape sheet not supplied Mix by pulse vortexing for 10 s Note Make sure that the punches are fully covered with buffer If necessary briefly centrifuge the S Block after mixing 44 BioSprint 96 DNA Handbook 06 2012 4 Place the S Block in a shaker incubator and incubate at 56 C with shaking at 900 rpm for 1 h 5 Towards the end of proteinase K digestion prepare five S Blocks slots 2 6 and two 96 well microplates slots 7 and 8 according to Table 11 below The S Blocks and microplates are loaded onto the worktable in step 15 In each plate or block the number of wells to be filled with buffer should match the number of samples to be processed e g if processing 48 samples fill 48 wells per plate or block Ensure that buffers are added to the same positions in each plate or block e g if processing 48 samples fill wells AI H1 to A6 H6 of each plate or block Table 11 BioSprint 96 worktable setup and reagent volumes Message Volume when per well Slot loading Plate block To add ul 8 Load 96 well Large 96 rod cover Rod Cover microplate MP 7 Load Elution 96 well Buffer AE 125 microplate MP 6 Load S Block RNase fre
58. ysis is usually complete in 1 3 h If it is more convenient samples can be lysed overnight this will not affect the DNA quality Optional If RNA free genomic DNA is required add 4 ul of RNase A 100 mg ml mix by vortexing and incubate for 2 min at room temperature 15 25 C 4 Towards the end of proteinase K digestion prepare five S Blocks slots 2 6 and two 96 well microplates slots 7 and 8 according to Table 9 on the next page The S Blocks and microplates are loaded onto the worktable in step 10 In each plate or block the number of wells to be filled with buffer should match the number of samples to be processed e g if processing 48 samples fill 48 wells per plate or block Ensure that buffers are added to the same positions in each plate or block e g if processing 48 samples fill wells AI H1 to A6 H6 of each plate or block BioSprint 96 DNA Handbook 06 2012 37 Table 9 BioSprint 96 worktable setup and reagent volumes Message Volume when per well Slot loading Plate block To add ul 8 Load 96 well Large 96 rod cover Rod Cover microplate MP 7 Load Elution 96 well Buffer AE 200 microplate MP 6 Load S Block RNase free water 500 Wash 5 5 Load S Block Buffer AW2 500 Wash 4 4 Load S Block Buffer AW2 500 Wash 3 3 Load S Block Buffer AW1 500 Wash 2 2 Load S Block Buffer AW1 650 Wash 1 1 Load Lysate S Block Lysatet 630 Contains 0 02 v v Tween 20 t Added at steps 1 2 and
Download Pdf Manuals
Related Search