Home

BioSprint 15 DNA Handbook

1. 13 Switch off the BioSprint 15 at the power switch 14 Wipe the surface of the tube strip tray and adjacent surfaces with a soft cloth or tissue moistened with distilled water or a mild detergent solution If infectious agents are spilt onto the tube strip tray clean using 70 ethanol or other disinfectant Note Do not use bleach as disinfectant See page 5 for safety information 26 BioSprint 15 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 15 workstation and the BioSprint 15 DNA Blood Kit Important points before starting M Check that QIAGEN Protease Buffer AW1 and Buffer AW2 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 15 Refer to the BioSprint 15 User Manual for operating instructions Things to do before starting E Set a water bath or shaker incubator to 70 C for use in step 8 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 lysate in step 5 of the procedure The final RNase A concentration should be 2 m
2. 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 A260 A320 x dilution factor Total amount of DNA isolated concentration x volume of sample in ml A260 As20 A2so A320 Purity of DNA sample 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 BioSprint 15 DNA Handbook 06 2012 49 Ordering Information Product BioSprint 15 DNA Blood Kit 45 BioSprint 15 DNA Blood Kit 360 Buffer ATL 200 ml QIAGEN Proteinase K 2 ml QIAGEN Proteinase K 10 ml Buffer AE 240 ml Accessories BioSprint 15 Plasticware 130 12 Tube Magnet Contents For 45 preps 5 Rod Covers 5 Tube Strips MagAttract Suspension G Buffers and Reagents For 360 preps 5 Rod Covers 5 Tube Strips 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 ml solution 240 ml Elution Buffer For 130 preps 26 x 5 Rod Cov
3. 15 After the protocol run ends press STOP slide out the tube strip tray and transfer the eluted DNA from well 5 of each 5 tube strip to other tubes for long term storage Note Well 5 is at the right of the 5 tube strip Carryover of magnetic particles in eluates will not affect most downstream applications If the risk of magnetic particle carryover needs to be minimized tubes containing eluate should first be placed in a suitable magnet and the eluates transferred to clean tubes see the appendix page 49 16 Remove the 5 tube strips and 5 rod covers and discard them according to your local safety regulations Note See page 5 for safety information 17 Switch off the BioSprint 15 at the power switch 18 Wipe the surface of the tube strip tray and adjacent surfaces with a soft cloth or tissue moistened with distilled water or a mild detergent solution If infectious agents are spilt onto the tube strip tray clean using 70 ethanol or other disinfectant Note Do not use bleach as disinfectant See page 5 for safety information 38 BioSprint 15 DNA Handbook 06 2012 Protocol Purification of DNA from Buccal Swabs This protocol is for purification of total genomic and mitochondrial DNA from buccal swabs using the BioSprint 15 workstation and the BioSprint 15 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
4. 15 DNA Handbook 06 2012 Well Reagent Volume of reagent pl Lysate 620 2 Buffer AW 700 3 Buffer AW2 500 4 Buffer AW2 500 5 Buffer AE 200 Added at steps 11 and 12 includes volume of Buffer ATL QIAGEN Proteinase K Buffer AL isopropanol and MagAttract Suspension G Note Well 1 is at the left of the 5 tube strip well 5 is at the right 9 Load up to three 5 rod covers into the rod cover slots There must always be a 5 rod cover above a column of 5 tube strips See page 17 for more information Insert a 5 rod cover into a rod cover slot so that the short tab faces inward and the long tab faces outward 5 rod covers must be inserted so that they click into place Note If necessary remove the tube strip tray to allow easier loading of the 5 rod covers Note Do not push 5 rod covers further after they click into place otherwise an instrument crash will occur 10 Briefly centrifuge the 2 ml microcentrifuge tube to remove drops from the inside of the lid 11 Carefully transfer 200 pl of the lysate to well 1 of the 5 tube strip leaving the swab in the 2 ml microcentrifuge tube Note Well 1 is at the left of the 5 tube strip well 5 is at the right Note If processing more than one sample record in which 5 tube strips you load the samples 12 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
5. 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 15 DNA procedure yields pure DNA with A240 Aaso 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 15 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 16 2 2 5 Heart 25 mg 5 9 2 6 Spleen 25 mg 69 1 23 6 Lung 25 mg 13 8 7 2 Liver 25 mg 77 8 29 4 Kidney 25 mg 26 2 18 8 Sheep tissue Ear 30 mg 20 3 1 8 Mouse tissue Tail 1 2 cm 25 mg 32 7 4 6 Cultured cells HL 60 2 x 10 cells 10 1 4 7 Blood Human 200 ul 4 5 9 0 5 7 x 10 cells ml Horse 200 ul 5 5 6 7 Bovine 200 ul 6 2 8 0 Sheep 200 ul 5 6 11 2 Pig 200 ul 4 5 9 0 Dog 200 ul 6 7 12 3 Cat 100 ul 4 4 8 3 Mouse 100 ul 2 0 8 0 Rat 100 ul 1 0 4 0 Bird 10 ul 15 0 6 3 Fish 5 ul 7 1 10 0 Genomic DNA was purified from the indicated samples Sample volume adjusted to 200 ul with Buffer AE Table 2 continued on next page BioSprint 15 DNA Handbook 06 2012 13 Table 2 continued from previous page Sample
6. 25 44 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 15 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 concentrate ml to add ml Final volume ml 17 AO 57 81 190 271 Tick the check box on the bottle to indicate that ethanol has been added Store reconstituted 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 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 A399 should be subtracted from all absorbance readings See the appendix page 49 for more information Loading 5 tube strip
7. MECL 1 was amplified using 5 ul purified DNA from the indicated cultured cell samples in a final reaction volume of 50 ul Amplification reactions were performed using the QIAGEN Taq PCR Core Kit A 5 ul aliquot of each PCR was run on a 1 5 agarose gel negative control positive control M 100 bp ladder BioSprint 15 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 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 concentrate ml to add ml Final volume ml 19
8. Sample volumes used in BioSprint 15 DNA procedures Sample Amount of sample Whole blood 100 300 ul Buffy coat 100 200 ul Cultured cells Up to 5x 10 diploid cells Tissues Up to 25 mg Rodent tails 1 2 cm approximately 25 mg Dried blood spots 1 3 punches 3 6 mm 1 8 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 pl with Buffer AE Storing blood samples Whole blood samples treated with EDTA ACD or heparin can 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 thi
9. buffy coat samples according to page 12 E Seta water bath or shaker incubator to 70 C for use in step 7 of the procedure E All samples in a single procedure must have the same volume 100 ul 200 ul or 300 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 Switch on the BioSprint 15 at the power switch 2 Open the front door of the BioSprint 15 and slide out the tube strip tray 3 Load up to fifteen 5 tube strips into the tube strip tray One 5 tube strip is used per sample If loading five 5 tube strips or fewer we recommend loading them as a single column If loading ten 5 tube strips or fewer we recommend loading them as 2 columns See page 17 for more information Load the 5 tube strips in the tube strip tray so that the tab of each 5 tube strip faces to the left Make sure that the 5 tube strips are fully inserted into the tray and are not skewed 4 Add reagents into each 5 tube strip according to the table on the next page 20 BioSprint 15 DNA Handbook 06 2012 Volume of reagent pl Well Reag
10. enzymatic downstream reactions Quantify the purified DNA by application spectrophotometric measurement of the absorbance at 260 nm see the appendix page 49 A2x60 A280 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 49 48 BioSprint 15 DNA Handbook 06 2012 Appendix Handling Q
11. is at the left of the 5 tube strip well 5 is at the right Note If processing more than one sample record in which 5 tube strips you load the samples 11 Vortex the master mix containing isopropanol and MagAttract Suspension G for 1 min see Things to do before starting Add 230 ul master mix to each sample in well 1 of each 5 tube strip Note If using a multidispenser add 225 ul master mix to each sample 12 Load up to three 5 rod covers into the rod cover slots There must always be a 5 rod cover above a column of 5 tube strips See page 17 for more information Insert a 5 rod cover into a rod cover slot so that the short tab faces inward and the long tab faces outward 5 rod covers must be inserted so that they click into place Note If necessary remove the tube strip tray to allow easier loading of the 5 rod covers Note Do not push 5 rod covers further after they click into place otherwi se an instrument crash will occur 13 Slide back the tube strip tray fully into the BioSprint 15 14 Close the front door of the BioSprint 15 Closing the front and top doors protects the samples from contamination 15 Select the protocol BS15 DNA Blood 200 using the A and VW keys on the BioSprint 15 workstation Press START to start the protocol run See the BioSprint 15 User Manual for safety information BioSprint 15 DNA Handbook 06 2012 29 16 After the protocol run ends press STO
12. or 300 ul This shorter protocol has less manual handling steps than the standard protocol see Protocol Purification of DNA from Blood Using the BioSprint 15 page 19 but yield and purity of the purified DNA may be lower Important points before starting E Check that QIAGEN Protease Buffer AW1 and Buffer AW2 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 HB This protocol is suitable for human whole blood Blood samples must be in the range of 100 300 ul E Ensure that you are familiar with operating the BioSprint 15 Refer to the BioSprint 15 User Manual for operating instructions E In some steps of the procedure one of three choices can be made Choose Ml if processing 100 ul blood samples choose A if processing 200 ul blood samples choose if processing 300 ul blood samples Things to do before starting E Thaw and equilibrate up to 15 whole blood samples at room temperature 15 25 C E All samples in a single procedure must have the same volume 100 ul 200 ul or 300 ul If the volume of a sample needs to be increased add the appropriate volume of PBS 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 before starting the procedure Th
13. power switch 18 Wipe the surface of the tube strip tray and adjacent surfaces with a soft cloth or tissue moistened with distilled water or a mild detergent solution If infectious agents are spilt onto the tube strip tray clean using 70 ethanol or other disinfectant Note Do not use bleach as disinfectant See page 5 for safety information 34 BioSprint 15 DNA Handbook 06 2012 Protocol Purification of DNA from Rodent Tails This protocol is for purification of total genomic and mitochondrial DNA from up to 1 2 cm approximately 25 mg of rodent tail per sample using the BioSprint 15 workstation and the BioSprint 15 DNA Blood Kit Important points before starting E Buffer ATL and QIAGEN Proteinase K are required for this protocol See Equipment and Reagents to Be Supplied by User page 9 M Check that Buffer AW1 and Buffer AW2 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 M Ensure that you are familiar with operating the BioSprint 15 Refer to the BioSprint 15 User Manual for operating instructions Things to do before starting M Seta shaker incubator to 56 C for use in step 3 of the procedure M MagAttract magnetic particles copurify RNA and DNA if both are present in the sample If RNA free DNA is requ
14. the 2 ml microcentrifuge tube and mix by pulse vortexing for 10 s 15 Briefly centrifuge the 2 ml microcentrifuge tube to remove drops from inside the lid 16 Carefully transfer the entire lysate from step 15 into well 1 of the 5 tube strip Note Well 1 is at the left of the 5 tube strip well 5 is at the right Note If processing more than one sample record in which 5 tube strips you load the samples 17 Add 20 pl MagAttract Suspension G to the lysate in well 1 of the 5 tube strip 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 18 Slide back the tube strip tray fully into the BioSprint 15 19 Close the front door of the BioSprint 15 Closing the front and top doors protects the samples from contamination 20 Select the protocol BS15 DNA Dried Blood using the A and VW keys on the BioSprint 15 workstation Press START to start the protocol run See the BioSprint 15 User Manual for safety information 21 After the protocol run ends press STOP slide out the tube strip tray and transfer the eluted DNA from well 5 of each 5 tube strip to other tubes for long term storage Note Well 5 is at the right of the 5 tube strip Carryover of magnetic particles in eluates will not affect most downstream applications If the risk of magnetic particle carryover needs to be minimized
15. 11 includes volume of sample QIAGEN Protease Buffer AL isopropanol and MagAttract Suspension G Note Well 1 is at the left of the 5 tube strip well 5 is at the right 5 Centrifuge the appropriate number of cells up to 5 x 106 for 5 min at 300 x g Discard the supernatant and resuspend the cell pellet in 200 pl PBS not supplied When using a frozen cell pellet allow cells to thaw until the pellet can be dislodged by gently flicking the tube before adding PBS Ensure that an appropriate number of cells is 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 6 Add 20 ul QIAGEN Protease to the sample 28 BioSprint 15 DNA Handbook 06 2012 7 Add 200 pl Buffer AL and 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 8 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 9 Briefly centrifuge the microcentrifuge tube to remove drops from the inside of the lid 10 Transfer the entire lysate to well 1 of the 5 tube strip Note Well 1
16. 31 Purification of DNA from Rodent Tails 35 E Purification of DNA from Buccal Swabs 39 a Purification of DNA from Dried Blood Spots 43 Troubleshooting Guide 47 Appendix Handling Quantification and Determination of Purity of DNA 49 Ordering Information 50 BioSprint 15 DNA Handbook 06 2012 3 Kit Contents BioSprint 15 DNA Blood Kit 45 360 Catalog no 940014 940017 Number of preps 45 360 Buffer ALt 12 ml 3x 33 ml QIAGEN Protease 1 vialt 2 vials Protease Solvent 1 2 ml 2x 4 4 ml MagAttract Suspension G 1 6 ml 13 ml Buffer AW1t concentrate 19 ml 1x19 ml 1 x 98 ml Buffer AW2 concentrate 17 ml 2x81 ml Buffer AE 15 ml 128 ml 5 Rod Cover 10 72 5 Tube Strip 1 ml 45 360 Quick Start Protocol 1 1 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 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 15 DNA Handbook 06 2012 Reconstituted QIAG
17. 52 BioSprint 15 DNA Handbook 06 2012 Notes BioSprint 15 DNA Handbook 06 2012 53 Notes 54 BioSprint 15 DNA Handbook 06 2012 Trademarks QIAGEN BioSprint MagAttract QIAGEN Group 903 FTA Whatman Whatman International Ltd DACRON INVISTA North America S A R L Corporation Eppendorf Eppendorf AG Finnpipette Thermo Electron Oy Corporation Puritan Puritan Medical Products Company Limited License Agreement for BioSprint 15 DNA 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 comp
18. BioSprint 15 DNA Blood Kit Human blood samples can be 100 ul 200 ul or 300 ul Animal blood samples containing non nucleated erythrocytes can be 100 ul or 200 ul Buffy coat samples can be 100 ul or 200 ul Important points before starting E Check that QIAGEN Protease Buffer AW1 and Buffer AW2 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 E Human blood samples must be in the range of 100 300 ul Animal blood samples must be in the range of 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 15 Refer to the BioSprint 15 User Manual for operating instructions E In some steps of the procedure one of three choices can be made Choose Ml if processing 100 ul blood samples choose A if processing 200 ul blood samples choose if processing 300 ul blood samples BioSprint 15 DNA Handbook 06 2012 19 Things to do before starting E Thaw and equilibrate up to 15 whole blood samples at room temperature 15 25 C or prepare
19. EN Protease 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 15 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 giagen 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 c
20. June 2012 BioSprint 15 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 15 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 Loading 5 tube strips and 5 rod covers into the BioSprint 15 17 Protocols Purification of DNA from Blood 19 Rapid Purification of DNA from Human Whole Blood 23 Purification of DNA from Cultured Cells 27 Purification of DNA from Tissues
21. 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 See Equipment and Reagents to Be Supplied by User page 9 M Check that Buffer AW1 and Buffer AW2 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 15 Refer to the BioSprint 15 User Manual for operating instructions Things to do before starting E Seta shaker incubator with an adapter for 2 ml microcentrifuge tubes to 56 C for use in step 4 of the procedure E Prepare a master mix according to the table below for use in step 12 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 Reagent Volume of reagent per sample ul Buffer AL 200 lsopropanol 200 MagAttract Suspension G 20 if BioSprint 15 DNA Handbook 06 2012 39 Procedure 1 Place the swab in a 2 ml microcentrifuge tube not supplied If using an Omni Swab eject the swa
22. P slide out the tube strip tray and transfer the eluted DNA from well 5 of each 5 tube strip to other tubes for long term storage Note Well 5 is at the right of the 5 tube strip Carryover of magnetic particles in eluates will not affect most downstream applications If the risk of magnetic particle carryover needs to be minimized tubes containing eluate should first be placed in a suitable magnet and the eluates transferred to clean tubes see the appendix page 49 17 Remove the 5 tube strips and 5 rod covers and discard them according to your local safety regulations Note See page 5 for safety information 18 Switch off the BioSprint 15 at the power switch 19 Wipe the surface of the tube strip tray and adjacent surfaces with a soft cloth or tissue moistened with distilled water or a mild detergent solution If infectious agents are spilt onto the tube strip tray clean using 70 ethanol or other disinfectant Note Do not use bleach as disinfectant See page 5 for safety information 30 BioSprint 15 DNA Handbook 06 2012 Protocol Purification of DNA from Tissues This protocol is for purification of total genomic and mitochondrial DNA from up to 25 mg of tissue per sample using the BioSprint 15 workstation and the BioSprint 15 DNA Blood Kit Important points before starting E Buffer ATL and QIAGEN Proteinase K are required for this protocol See Equipment and Reagents to Be Supplied by User page 9 M C
23. Procedure 1 Cut 3 mm 1 8 inch or 6 mm 1 4 inch diameter punches from a dried blood spot with a single hole paper punch Place up to 3 blood card punches into a 2 ml microcentrifuge tube not supplied 2 Add 200 pl Buffer ATL not supplied 3 Add 20 ul QIAGEN Proteinase K not supplied close the 2 ml microcentrifuge tube and mix thoroughly by pulse vortexing for 10s Note Make sure that the punches are fully covered with buffer If necessary briefly centrifuge the 2 ml microcentrifuge tube 4 Place the 2 ml microcentrifuge tube 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 switch on the BioSprint 15 at the power switch 6 Open the front door of the BioSprint 15 and slide out the tube strip tray BioSprint 15 DNA Handbook 06 2012 43 7 Load up to fifteen 5 tube strips into the tube strip tray One 5 tube 10 strip is used per sample If loading five 5 tube strips or fewer we recommend loading them as a single column If loading ten 5 tube strips or fewer we recommend loading them as 2 columns See page 17 for more information Load the 5 tube strips in the tube strip tray so that the tab of each 5 tube strip faces to the left Make sure that the 5 tube strips are fully inserted into the tray and are not skewed Add reagents into each 5 tube strip according to the table below Well Reagent Volume of reagent pl Lysa
24. b 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 2 ml microcentrifuge tube If using a cotton or Dacron swab add 400 ul Buffer ATL to the 2 ml microcentrifuge tube 3 Add 20 pl QIAGEN Proteinase K not supplied close the 2 ml microcentrifuge tube and mix thoroughly by pulse vortexing for 10s 4 Place the 2 ml microcentrifuge tube 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 5 Towards the end of proteinase K digestion switch on the BioSprint 15 at the power switch 6 Open the front door of the BioSprint 15 and slide out the tube strip tray 7 Load up to fifteen 5 tube strips into the tube strip tray One 5 tube strip is used per sample If loading five 5 tube strips or fewer we recommend loading them as a single column If loading ten 5 tube strips or fewer we recommend loading them as 2 columns See page 17 for more information Load the 5 tube strips in the tube strip tray so that the tab of each 5 tube strip faces to the left Make sure that the 5 tube strips are fully inserted into the tray and are not skewed 8 Add reagents into each 5 tube strip according to the table on the next page 40 BioSprint
25. blood spots M Microcentrifuge tubes 1 5 ml required for lysis steps when processing blood not needed for rapid blood protocol cells tissues or rodent tails E Microcentrifuge tubes 2 ml required for lysis steps when processing buffy coat swabs or dried blood spots E Microcentrifuge not needed for rapid blood protocol 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 15 DNA Handbook 06 2012 9 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 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 15 workstation E Manual paper punch 3 6 mm 1 8 1 4 inch if processing dried blood spots This is not a complete list of suppliers and does not include many important vendors of biological supplies 10 BioSprint 15 DNA Handbook 06 2012 Important Notes Starting material The amounts of starting material for use in BioSprint 15 DNA procedures are shown in Table 1 Table 1
26. e 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 M 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 BioSprint 15 DNA Handbook 06 2012 23 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 A 450 ul or 650 ul master mix is required per sample see step 6 of the protocol The starting volume of master mix should be increased accordingly Volume of reagent per sample ul Reagent a A Buffer AL 100 200 300 lsopropanol 100 200 300 MagAttract Suspension G 15 30 45 Procedure 1 Switch on the BioSprint 15 at the power switch 2 Open the front door of the BioSprint 15 and slide out the tube strip tray 3 Load up to fifteen 5 tube strips into the tube strip tray One 5 tube strip is used per sample If loading five 5 tube strips or fewer we recommend loading them as a single column If loading ten 5 tube strips or fewer we recommend loading them as 2 columns See page 17 for more information Load the 5 tube strips in the tube strip tray so that the tab of each 5 tube strip faces to the left Make sure that the 5 tube str
27. each sample in well 1 of each 5 tube strip Note If using a multidispenser add 400 ul master mix to each sample 13 Slide back the tube strip tray fully into the BioSprint 15 14 Close the front door of the BioSprint 15 Closing the front and top doors protects the samples from contamination BioSprint 15 DNA Handbook 06 2012 41 15 Select the protocol BS15 DNA Swab using the A and VW keys on the BioSprint 15 workstation Press START to start the protocol run See the BioSprint 15 User Manual for safety information 16 After the protocol run ends press STOP slide out the tube strip tray and transfer the eluted DNA from well 5 of each 5 tube strip to other tubes for long term storage Note Well 5 is at the right of the 5 tube strip Carryover of magnetic particles in eluates will not affect most downstream applications If the risk of magnetic particle carryover needs to be minimized tubes containing eluate should first be placed in a suitable magnet and the eluates transferred to clean tubes see the appendix page 49 17 Remove the 5 tube strips and 5 rod covers and discard them according to your local safety regulations Note See page 5 for safety information 18 Switch off the BioSprint 15 at the power switch 19 Wipe the surface of the tube strip tray and adjacent surfaces with a soft cloth or tissue moistened with distilled water or a mild detergent solution If infectious agents are spi
28. ent m A 1 Lysate 325 650 975 2 Buffer AW 500 700 1000 3 Buffer AW2 500 500 500 4 Buffer AW2 500 500 500 5 Buffer AE 100 200 300 Added at steps 11 and 12 includes volume of sample QIAGEN Protease Buffer AL isopropanol and MagAttract Suspension G Note Well 1 is at the left of the 5 tube strip well 5 is at the right 5 Pipet W 10 ul A 20 ul or 30 ul QIAGEN Protease into the bottom of a microcentrifuge tube not supplied use a 1 5 ml tube for blood samples and a 2 ml tube for buffy coat samples Add W 100 ul A 200 ul or 300 ul sample to the QIAGEN Protease Note It is possible to add QIAGEN Protease to samples that have already been dispensed into microcentrifuge tubes In this case it is important to ensure proper mixing after adding QIAGEN Protease 6 Add 100 ul A 200 ul or 300 ul Buffer AL and 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 homogeneous solution Note Do not add QIAGEN Protease directly to Buffer AL 7 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 8 Briefly centrifuge the microcentrifuge tube to remove drops from the inside of the lid 9 Add W 100 ul A 200 ul or 300 pl isopropanol and mix by pulse vortexing for 10 s 10 Briefly centrifuge the microcentrifuge tube to remove d
29. er AL immediately and thoroughly by Buffer AL pulse vortexing 2 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 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 BioSprint 15 DNA Handbook 06 2012 47 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 49 b Excess DNA used in Excess DNA can inhibit some
30. ers and 130 x 5 Tube Strips for use with the BioSprint 15 Magnet for separating magnetic particles in 12 x 1 5 ml or 2 ml tubes BioSprint 96 DNA Blood Kit for rapid purification of DNA from cells tissue blood buffy coat buccal swabs and dried blood spots using the BioSprint 96 workstation BioSprint 96 DNA Blood Kit 48 For 48 preps Large 96 Rod Covers 96 Well Microplates MP S Blocks MagAttract Suspension G Buffers and Reagents Other kit sizes are available see www qiagen com 50 Cat no 940014 940017 19076 19131 19133 19077 1030058 36912 940054 BioSprint 15 DNA Handbook 06 2012 Product Contents Cat no 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 941517 Kit 360 Strips MagAttract Suspension G Buffers and Reagents BioSprint 96 DNA Plant For 576 preps Large 96 Rod Covers 941557 Kit 576 96 Well Microplates MP S Blocks MagAttract Suspension G Buffers and Reagents 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 Other kit sizes are available see www giagen com i E BioSprint 15 DNA Handbook 06 2012 51 Notes
31. g 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 below for use in step 11 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 wl lsopropanol 200 MagAttract Suspension G 30 BioSprint 15 DNA Handbook 06 2012 27 Procedure 1 Switch on the BioSprint 15 at the power switch 2 Open the front door of the BioSprint 15 and slide out the tube strip tray 3 Load up to fifteen 5 tube strips into the tube strip tray One 5 tube strip is used per sample If loading five 5 tube strips or fewer we recommend loading them as a single column If loading ten 5 tube strips or fewer we recommend loading them as 2 columns See page 17 for more information Load the 5 tube strips in the tube strip tray so that the tab of each 5 tube strip faces to the left Make sure that the 5 tube strips are fully inserted into the tray and are not skewed 4 Add reagents into each 5 tube strip according to the table below Well Reagent Volume of reagent pl Lysate 650 2 Buffer AW1 700 3 Buffer AW2 500 4 Buffer AW2 500 5 Buffer AE 200 Added at steps 10 and
32. heck that Buffer AW1 and Buffer AW2 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 15 Refer to the BioSprint 15 User Manual for operating instructions Things to do before starting M Set a shaker incubator 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 lysate 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 Prepare a master mix according to the table on the next page for use in step 10 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 10 of the procedure The starting volume of master mix should be increased accordingly E aa BioSprint 15 DNA Handbook 06 2012 31 Reagent Volume of reagent per
33. hort tab faces inward and the long tab faces outward 5 rod covers must be inserted so that they click into place Note If necessary remove the tube strip tray to allow easier loading of the 5 rod covers Note Do not push 5 rod covers further after they click into place otherwise an instrument crash will occur 12 Slide back the tube strip tray fully into the BioSprint 15 13 Close the front door of the BioSprint 15 Closing the front and top doors protects the samples from contamination BioSprint 15 DNA Handbook 06 2012 33 14 Select the protocol BS15 DNA Tissue using the A and V keys on the BioSprint 15 workstation Press START to start the protocol run See the BioSprint 15 User Manual for safety information 15 After the protocol run ends press STOP slide out the tube strip tray and transfer the eluted DNA from well 5 of each 5 tube strip to other tubes for long term storage Note Well 5 is at the right of the 5 tube strip Carryover of magnetic particles in eluates will not affect most downstream applications If the risk of magnetic particle carryover needs to be minimized tubes containing eluate should first be placed in a suitable magnet and the eluates transferred to clean tubes see the appendix page 49 16 Remove the 5 tube strips and 5 rod covers and discard them according to your local safety regulations Note See page 5 for safety information 17 Switch off the BioSprint 15 at the
34. in the tube strip tray so that the tab of each 5 tube strip faces to the left Make sure that the 5 tube strips are fully inserted into the tray and are not skewed Add reagents into each 5 tube strip according to the table on the next page BioSprint 15 DNA Handbook 06 2012 Well Reagent Volume of reagent pl Lysate 630 2 Buffer AW 700 3 Buffer AW2 500 4 Buffer AW2 500 5 Buffer AE 200 Added at steps 9 and 10 includes volume of sample Buffer ATL QIAGEN Proteinase K Buffer AL isopropanol and MagAttract Suspension G Note Well 1 is at the left of the 5 tube strip well 5 is at the right 8 Briefly centrifuge the 1 5 ml microcentrifuge tube to remove drops from the inside of the lid 9 Transfer the entire lysate to well 1 of the 5 tube strip Note Well 1 is at the left of the 5 tube strip well 5 is at the right Note If processing more than one sample record in which 5 tube strips you load the samples 10 Vortex the master mix containing Buffer AL isopropanol and MagAttract Suspension G for 1 min see Things to do before starting Add 430 pl of master mix to each sample in well 1 of each 5 tube strip Note If using a multidispenser add 450 ul master mix to each sample 11 Load up to three 5 rod covers into the rod cover slots There must always be a 5 rod cover above a column of 5 tube strips See page 17 for more information Insert a 5 rod cover into a rod cover slot so that the s
35. ips are fully inserted into the tray and are not skewed 4 Add reagents into each 5 tube strip according to the table on the next page 24 BioSprint 15 DNA Handbook 06 2012 Volume of reagent ul Well Reagent m A 1 Lysate 325 650 975 2 Buffer AW1 500 700 1000 3 Buffer AW2 500 500 500 4 Buffer AW2 500 500 500 5 Buffer AE 100 200 300 Added at steps 5 and 6 includes volume of sample QIAGEN Protease Buffer AL isopropanol and MagAttract Suspension G Note Well 1 is at the left of the 5 tube strip well 5 is at the right 5 Pipet W 10 ul A 20 ul or 30 ul QIAGEN Protease into the bottom of well 1 of each 5 tube strip Add W 100 ul A 200 ul or 300 ul sample to the QIAGEN Protease Note Well 1 is at the left of the 5 tube strip well 5 is at the right Note If processing more than one sample record in which 5 tube strips you load the samples 6 Vortex the master mix containing Buffer AL isopropanol and MagAttract Suspension G for 1 min see Things to do before starting Add W 215 ul A 430 ul or 645 ul master mix to each sample in well 1 of each 5 tube strip Note If using a multidispenser add W 225 ul A 450 ul or 650 ul master mix to each sample 7 Load up to three 5 rod covers into the rod cover slots There must always be a 5 rod cover above a column of 5 tube strips See page 17 for more information Insert a 5 rod cover into a rod cover slot so that the sho
36. ired 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 Prepare a master mix according to the table on the next page for use in step 10 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 10 of the procedure The starting volume of master mix should be increased accordingly aaa a BioSprint 15 DNA Handbook 06 2012 35 Reagent Volume of reagent per sample ul Buffer AL 200 lsopropanol 200 MagAttract Suspension G 30 Procedure 1 Cut 1 2 cm of each rodent tail sample into small pieces Place the tissue sample 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 close the 1 5 ml microcentrifuge tube 3 Place the 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
37. is Pure DNA Rapid protocol only available for blood BioSprint 15 DNA Handbook 06 2012 uoypindaid ajduins jpnupw uoypoyLind WNq wous pajpwoynn Ajjn4 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 15 workstation cat no 9000850 E Pipettors and disposable pipet tips with aerosol barriers 20 1000 ul M Multidispenser e g Finnpipette Stepper from Thermo Electron see www thermo com HE Water bath or a shaker incubator not required for rapid blood protocol e g Eppendorf Thermomixer Comfort cat no 5355 000 011 M 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 M Disposable gloves E Vortexer M Soft cloth or tissue and 70 ethanol or other disinfectant to clean the BioSprint worktable Buffer ATL cat no 19076 if processing tissues rodent tails swabs or 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 or dried blood spots E DNase free RNase A required if purified DNA needs to be RNA free not required if processing swabs or dried
38. le and procedure The BioSprint 15 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 15 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 Sa E BioSprint 15 DNA Handbook 06 2012 7 BioSprint 15 DNA Procedure Standard protocol Rapid blood protocol Sample Lysis Add isopropanol and MagAttract Suspension G Add sample Transfer to and reagents 5 tube strip to 5 tube strip DNA binds to magnetic particles Magnetic separation Wash Magnetic separation Elute un lt G
39. lean with suitable laboratory detergent and water If the spilt liquid contains potentially infectious agents clean the affected area first with laboratory detergent and water and then with 1 v v sodium hypochlorite If liquid containing potentially infectious agents is spilt on the BioSprint 15 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 15 DNA Handbook 06 2012 5 Quality Control In accordance with QIAGEN s ISO certified Quality Management System each lot of BioSprint 15 DNA Blood Kit is tested against predetermined specifications to ensure consistent product quality 6 BioSprint 15 DNA Handbook 06 2012 Introduction The BioSprint 15 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 samples using the BioSprint 15 workstation The BioSprint 15 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 Princip
40. ll 1 of the 5 tube strip Note Well 1 is at the left of the 5 tube strip well 5 is at the right Note If processing more than one sample record in which 5 tube strips you load the samples 10 Vortex the master mix containing Buffer AL isopropanol and MagAttract Suspension G for 1 min see Things to do before starting Add 430 pl of master mix to each sample in well 1 of each 5 tube strip Note If using a multidispenser add 450 ul master mix to each sample 11 Load up to three 5 rod covers into the rod cover slots There must always be a 5 rod cover above a column of 5 tube strips See page 17 for more information Insert a 5 rod cover into a rod cover slot so that the short tab faces inward and the long tab faces outward 5 rod covers must be inserted so that they click into place Note If necessary remove the tube strip tray to allow easier loading of the 5 rod covers Note Do not push 5 rod covers further after they click into place otherwise an instrument crash will occur 12 Slide back the tube strip tray fully into the BioSprint 15 13 Close the front door of the BioSprint 15 Closing the front and top doors protects the samples from contamination BioSprint 15 DNA Handbook 06 2012 37 14 Select the protocol BS15 DNA Tissue using the A and V keys on the BioSprint 15 workstation Press START to start the protocol run See the BioSprint 15 User Manual for safety information
41. lt onto the tube strip tray clean using 70 ethanol or other disinfectant Note Do not use bleach as disinfectant See page 5 for safety information 42 BioSprint 15 DNA Handbook 06 2012 Protocol Purification of DNA from Dried Blood Spots This protocol is for purification of total genomic and mitochondrial DNA from blood card punches using the BioSprint 15 workstation and the BioSprint 15 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 15 Important points before starting E Buffer ATL and QIAGEN Proteinase K are required for this protocol See Equipment and Reagents to Be Supplied by User page 9 M Check that Buffer AW1 and Buffer AW2 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 15 Refer to the BioSprint 15 User Manual for operating instructions Things to do before starting E Set a shaker incubator with an adapter for 2 ml microcentrifuge tubes to 56 C for use in steps 4 and 12 of the procedure
42. 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 switch on the BioSprint 15 at the power switch 5 Open the front door of the BioSprint 15 and slide out the tube strip tray 6 Load up to fifteen 5 tube strips into the tube strip tray One 5 tube strip is used per sample If loading five 5 tube strips or fewer we recommend loading them as a single column If loading ten 5 tube strips or fewer we recommend loading them as 2 columns See page 17 for more information Load the 5 tube strips in the tube strip tray so that the tab of each 5 tube strip faces to the left Make sure that the 5 tube strips are fully inserted into the tray and are not skewed 36 BioSprint 15 DNA Handbook 06 2012 7 Add reagents into each 5 tube strip according to the table below Well Reagent Volume of reagent pl Lysate 630 2 Buffer AW 700 3 Buffer AW2 500 4 Buffer AW2 500 5 Buffer AE 200 Added at steps 9 and 10 includes volume of sample Buffer ATL QIAGEN Proteinase K Buffer AL isopropanol and MagAttract Suspension G Note Well 1 is at the left of the 5 tube strip well 5 is at the right 8 Briefly centrifuge the 1 5 ml microcentrifuge tube to remove drops from the inside of the lid 9 Transfer the entire lysate to we
43. nl 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 pane Sample amp Assay Technologies
44. o start the protocol run See the BioSprint 15 User Manual for safety information 17 After the protocol run ends press STOP slide out the tube strip tray and transfer the eluted DNA from well 5 of each 5 tube strip to other tubes for long term storage Note Well 5 is at the right of the 5 tube strip Carryover of magnetic particles in eluates will not affect most downstream applications If the risk of magnetic particle carryover needs to be minimized tubes containing eluate should first be placed in a suitable magnet and the eluates transferred to clean tubes see the appendix page 49 18 Remove the 5 tube strips and 5 rod covers and discard them according to your local safety regulations Note See page 5 for safety information 19 Switch off the BioSprint 15 at the power switch 20 Wipe the surface of the tube strip tray and adjacent surfaces with a soft cloth or tissue moistened with distilled water or a mild detergent solution If infectious agents are spilt onto the tube strip tray clean using 70 ethanol or other disinfectant Note Do not use bleach as disinfectant See page 5 for safety information 22 BioSprint 15 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 15 workstation and the BioSprint 15 DNA Blood Kit Blood samples can be 100 ul 200 ul
45. onents 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 Or 3 SND 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 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 qiagen 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 b
46. rops from the inside of the lid 11 Transfer the entire lysate into well 1 of the 5 tube strip Note Well 1 is at the left of the 5 tube strip well 5 is at the right Note If processing more than one sample record in which 5 tube strips you load the samples BioSprint 15 DNA Handbook 06 2012 21 12 Add 15 ul A 30 ul or 45 ul MagAttract Suspension G to the lysate in well 1 of the 5 tube strip 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 13 Load up to three 5 rod covers into the rod cover slots There must always be a 5 rod cover above a column of 5 tube strips See page 17 for more information Insert a 5 rod cover into a rod cover slot so that the short tab faces inward and the long tab faces outward 5 rod covers must be inserted so that they click into place Note If necessary remove the tube strip tray to allow easier loading of the 5 rod covers Note Do not push 5 rod covers further after they click into place otherwise an instrument crash will occur 14 Slide back the tube strip tray fully into the BioSprint 15 15 Close the front door of the BioSprint 15 Closing the front and top doors protects the samples from contamination 16 Select the protocol W BS15 DNA Blood 100 A BS15 DNA Blood 200 or BS15 DNA Blood 300 using the A and VW keys Press START t
47. rt tab faces inward and the long tab faces outward 5 rod covers must be inserted so that they click into place Note If necessary remove the tube strip tray to allow easier loading of the 5 rod covers Note Do not push 5 rod covers further after they click into place otherwise an instrument crash will occur 8 Slide back the tube strip tray fully into the BioSprint 15 BioSprint 15 DNA Handbook 06 2012 25 9 Close the front door of the BioSprint 15 Closing the front and top doors protects the samples from contamination 10 Select the protocol W BS15 DNA Blood 100 A BS15 DNA Blood 200 or BS15 DNA Blood 300 using the A and VW keys Press START to start the protocol run See the BioSprint 15 User Manual for safety information 11 After the protocol run ends press STOP slide out the tube strip tray and transfer the eluted DNA from well 5 of each 5 tube strip to other tubes for long term storage Note Well 5 is at the right of the 5 tube strip Carryover of magnetic particles in eluates will not affect most downstream applications If the risk of magnetic particle carryover needs to be minimized tubes containing eluate should first be placed in a suitable magnet and the eluates transferred to clean tubes see the appendix page 49 12 Remove the 5 tube strips and 5 rod covers and discard them according to your local safety regulations Note See page 5 for safety information
48. s and 5 rod covers into the BioSprint 15 Up to fifteen 5 tube strips can be loaded into the tube strip tray One 5 tube strip is used per sample If loading five 5 tube strips or fewer we recommend loading them as a single column If loading ten 5 tube strips or fewer we recommend loading them as 2 columns 5 tube strips are loaded into the tube strip tray so that the tab of each 5 tube strip faces to the left The 5 tube strips should be fully inserted into the tray and not skewed BioSprint 15 DNA Handbook 06 2012 17 Tab 00009 00000 00000 2000 0000o 00000 Tab Left column Middle column Right column Figure 4 Correct loading of 5 tube strips in the tube strip tray Up to three 5 rod covers can be loaded into the rod cover slots There must always be a 5 rod cover above a column of 5 tube strips Rod cover slot Figure 5 Rod cover slot Insert a 5 rod cover into a rod cover slot so that the short tab faces inward and the long tab faces outward 5 rod covers must be inserted so that they click into place Short tab Long tab Figure 6 Tabs of the 5 rod cover Note Do not push 5 rod covers further after they click into place otherwise an instrument crash will occur 18 BioSprint 15 DNA Handbook 06 2012 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 15 workstation and the
49. s 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 15 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 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
50. sample ul Buffer AL 200 lsopropanol 200 MagAttract Suspension G 30 Procedure 1 32 Cut lt 25 mg of each tissue sample into small pieces Place a tissue sample into a 1 5 ml microcentrifuge tube not supplied and add 180 pl Buffer ATL not supplied Add 20 ul QIAGEN Proteinase K not supplied and close the 1 5 ml microcentrifuge tube Place the 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 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 Towards the end of proteinase K digestion switch on the BioSprint 15 at the power switch Open the front door of the BioSprint 15 and slide out the tube strip tray Load up to fifteen 5 tube strips into the tube strip tray One 5 tube strip is used per sample If loading five 5 tube strips or fewer we recommend loading them as a single column If loading ten 5 tube strips or fewer we recommend loading them as 2 columns See page 17 for more information Load the 5 tube strips
51. te 640 2 Buffer AW 700 3 Buffer AW2 500 4 Buffer AW2 500 5 Buffer AE 125 Added at steps 16 and 17 includes the volume of Buffer ATL QIAGEN Proteinase K Buffer AL isopropanol and MagAttract Suspension G Note Well 1 is at the left of the 5 tube strip well 5 is at the right Load up to three 5 rod covers into the rod cover slots There must always be a 5 rod cover above a column of 5 tube strips See page 17 for more information Insert a 5 rod cover into a rod cover slot so that the short tab faces inward and the long tab faces outward 5 rod covers must be inserted so that they click into place Note If necessary remove the tube strip tray to allow easier loading of the 5 rod covers Note Do not push 5 rod covers further after they click into place otherwise an instrument crash will occur Briefly centrifuge the 2 ml microcentrifuge tube to remove drops from the inside of the lid 11 Add 200 ul Buffer AL close the 2 ml microcentrifuge tube and mix 44 by pulse vortexing for 10 s Note Make sure that the punches are fully covered with buffer If necessary briefly centrifuge the 2 ml microcentrifuge tube BioSprint 15 DNA Handbook 06 2012 12 Place the 2 ml microcentrifuge tube in a shaker incubator and incubate at 56 C with shaking at 900 rpm for 10 min 13 Briefly centrifuge the 2 ml microcentrifuge tube to remove drops from inside the lid 14 Add 200 ul isopropanol close the lid of
52. tubes containing eluate should first be placed in a suitable magnet and the eluates transferred to clean tubes see the appendix page 49 22 Remove the 5 tube strips and 5 rod covers and discard them according to your local safety regulations Note See page 5 for safety information BioSprint 15 DNA Handbook 06 2012 45 23 Switch off the BioSprint 15 at the power switch 24 Wipe the surface of the tube strip tray and adjacent surfaces with a soft cloth or tissue moistened with distilled water or a mild detergent solution If infectious agents are spilt onto the tube strip tray clean using 70 ethanol or other disinfectant Note Do not use bleach as disinfectant See page 5 for safety information 46 BioSprint 15 DNA Handbook 06 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 giagen com FAQ FAQList aspx The scientists in QIAGEN Technical Services are always happy to answer any questions you may have about either the information and protocols in this handbook or sample and assay technologies for contact information see back cover or visit www giagen com 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 Buff
53. type Amount of sample Typical DNA yield ug Dried blood spots 903 Specimen 1 punch 6 mm 1 4 inch 0 30 Collection Paper diameter FTA Card 1 punch 6 mm 1 4 inch 0 12 diameter Swabs Buccal swabs 1 swab 0 8 2 0 Genomic DNA was purified from the indicated samples b s 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 15 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 M markers Lambda Hindlll 14 BioSprint 15 DNA Handbook 06 2012 M Sheep ear tissue M lt 0 oe oe Oe Figure 2 Reproducible purification of genomic DNA from sheep ear tissue Sheep ear tissue 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 15 DNA Blood Kit with the BioSprint 15 DNA Tissue protocol DNA was eluted in 200 pl elution buffer Eluates 5 ul were run on a 0 8 agarose gel in 1x TBE M markers Lambda Hindill M BJAB Hela S3 HL 60 Ned ese ee ees ke Figure 3 Efficient amplification of the single copy gene MECL 1 The single copy gene
54. uantification and Determination of Purity of DNA Storage of DNA Purified DNA may be stored at 2 8 C for 24 hours 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 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 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 Tris HCl pH 7 5 to dilute DNA samples and to calibrate the spectrophotometer Pure DNA has an Aj60 Azg9 ratio of 1 7 1 9

BioSprint 15 DNA Handbook

Contents

Download Pdf Manuals

Related Search

Related Contents