Genomic DNA from Blood
Contents
1. 35 60 85 110 135 160 185 Recovered elution buffer Lil Elution of genomic DNA vacuum processing 100 Recovery Total DNA yield recovery m and concentration of recovered DNA e are plotted versus dispensed elution buffer volume High elution buffer volumes result in high elu tion efficiency whereas high concentrated DNA solutions can be obtained with smaller elution buffer volumes The dead volume of the silica membrane under vacuum is ap proximatively 15 ul 12 MACHEREY NAGEL 02 2009 Rev 05 Genomic DNA from Blood 2 4 Automated processing on robotic platforms NucleoSpin 8 96 Blood can be fully automated on many common laboratory work stations For the availability of scripts and general considerations about adapting NucleoSpin 8 96 Blood on a certain workstation please contact MN Full processing under vacuum enables complete automation without the need for centrifugation steps for drying of the membrane or for elution The risk of cross contamination is reduced by optimized vacuum settings during the elution step and by the improved shape of the outlets of the NucleoSpin 8 96 Blood Binding Strips Plate Drying of the NucleoSpin 8 96 Blood Binding Strips Plate under vacuum is suf ficient because the bottom of the strips plate is protected from spraying wash buffer during the washing steps by the MN Wash Plate Thus if possible the MN Wash Plate should be integrated i2. S 22 S24 S 25 S 26 S 36 37 Keep container tightly closed Keep away from sources of ignition No smoking Do not breathe dust Avoid contact with the skin Avoid contact with the eyes In case of contact with eyes rinse immediately with plenty of water and seek medical advice Wear suitable protective clothing and gloves Hazard labeling not neccessary if quantity per bottle below 125g or ml certificate of exemption according to 67 548 EEC Art 25 1999 45 EC Art 12 and German GefStoffV 20 3 and TRGS 200 7 1 For further information see Material Safety Data Sheet 16 MACHEREY NAGEL 02 2009 Rev 05 NucleoSpin 8 Blood 5 General procedure NucleoSpin 8 Blood vacuum processing For details on each step see section 5 1 Before starting the preparation e Check if Wash Buffer B5 and Proteinase K were prepared according to sec tion 3 1 Lyse samples 200 pl blood equilibrated to room temperature 25 yl Proteinase K 200 yl BQ1 Mix 3 times Incubate at RT 10 min or Mix 3 times and shake at e g Rack of Tube Strips 1250 rpm at RT with Cap Strips 10 min not supplied with the kit 2 Adjust DNA binding 200 pl ethanol conditions Mix at least 3 5 times Note High speed pipetting 400 ul s should be used for optimized mixing if possible 3 Load samples Transfer samples to NucleoSpin Blood Binding Strips 4 Overlay with Buffer B5 150 pl B5 5 BRE to si
3. consequential or spe cial including but not limited to loss of use revenue or profit whether based upon warranty contract tort including negligence or strict liability arising in connection with the sale or the failure of MACHEREY NAGEL products to perform in accordance with the stated specifications This warranty is exclusive and MACHEREY NAGEL makes no other warranty expressed or implied The warranty provided herein and the data specifications and descriptions of this MACHEREY NAGEL product appearing in MACHEREY NAGEL published catalogues and product literature are MACHEREY NAGEL s sole representations concerning 34 MACHEREY NAGEL 02 2009 Rev 05 Genomic DNA from Blood the product and warranty No other statements or representations written or oral by MACHEREY NAGEL s employees agent or representatives except written state ments signed by a duly authorized officer of MACHEREY NAGEL are authorized they should not be relied upon by the customer and are not a part of the contract of sale or of this warranty Product claims are subject to change Therefore please contact our Technical Service Team for the most up to date information on MACHEREY NAGEL products You may also contact your local distributor for general scientific information Applications mentioned in MACHEREY NAGEL literature are provided for informational purposes only MACHEREY NAGEL does not warrant that all applications have been tested in MACHEREY N
4. Binding Plate and MN Wash Plate MACHEREY NAGEL 02 2009 Rev 05 19 NucleoSpin 96 Blood 5 Bind DNA to silica mem 0 2 bar brane 5 min 6 Wash silica membrane 600 ul BW 0 2 bar 3 min 900 ul BS 0 2 bar 1 min NucleoSpin Blood Binding Plate and MN Wash Plate 900 ul B5 0 2 bar 1 min 7 Dry silica membrane Remove MN Wash Plate 0 6 bar 10 min 8 Elute DNA 50 200 ul BE Incubate 5 min 0 6 bar 1 min NucleoSpin Blood Binding Plate on Rack of Tube Strips Reduction of atmospheric pressure 20 MACHEREY NAGEL 02 2009 Rev 05 NucleoSpin 8 Blood 5 1 NucleoSpin 8 Blood vacuum processing For processing of NucleoSpin 8 Blood under vacuum the NucleoVac 96 Vacuum Manifold and the Starter Kit A are required see ordering information Starter Kit A con tains the Column Holders A and the NucleoSpin Dummy Strips to close unused rows of the Column Holder A Alternatively other suitable vacuum manifolds can be used The use of NucleoSpin Blood Binding Strips in a Column Holder A allows the isolation of up to n x 8 samples n 1 to 6 Insert as many of the NucleoSpin Blood Binding Strips as required into the reusable column holder and place it onto an MN Square well Block Before starting the preparation e Check if Wash Buffer B5 and Proteinase K were prepared according to sec tion 3 Lysis tubes are not supplied with t
5. a maximum of 1 year Storage at lower temperatures may cause precipitation of salts If a salt precipitation is observed incubate the bottle at 30 40 C for some minutes and mix well until all of the precipitate is redissolved Before starting with any NucleoSpin 8 96 Blood kit procedure prepare the following e Wash Buffer B5 Add the indicated volume of 96 100 ethanol to the Buffer B5 Concentrate Mark the label of the bottle to indicate that ethanol is added Store Wash Buffer B5 at room temperature 20 25 C for up to one year Before first use of the kit add the indicated volume of Proteinase Buffer to ly ophilized Proteinase K Proteinase K solution is stable at 20 C for 6 months NucleoSpin 8 Blood 12 x 8 preps 60 x 8 preps Cat No 740664 740664 5 Wash Buffer B5 100 ml 5 x 100 ml Concentrate Add 400 ml ethanol Add 400 ml ethanol to each bottle Proteinase K 75 mg 5x 75mg lyophilized Add 3 35 ml Proteinase Buffer Add 3 35 ml Proteinase Buffer to each bottle 14 MACHEREY NAGEL 02 2009 Rev 05 Genomic DNA from Blood NucleoSpin 96 Blood 1 x 96 preps 4 x 96 preps 24 x 96 preps Cat No 740665 1 740665 4 740665 24 Wash Buffer B5 100 ml 4x 100 ml 24 x 100 ml Concentrate Add 400 ml ethanol Add 400 ml ethanol Add 400 ml ethanol to each bottle to each bottle Proteinase K 75 mg 4 x 75 mg 24 x 75 mg lyophilized Add 3 35 ml Add 3 35 ml Add 3 35 Proteinase Buffer Proteinase Buffer Prote
6. clean paper towel to remove residual EtOH containing wash buffer Remove manifold lid MN Wash Plate and waste container from the vacuum manifold Dry silica membrane Remove any residual washing buffer from the outlets of the NucleoSpin Blood Binding Strips If necessary tap the outlets onto a clean paper sheet sup plied with the MN Wash Plate or soft tissue until no drops come out Insert the Column Holder A with the NucleoSpin Blood Binding Strips again into the lid and close the manifold Apply maximum vacuum at least 0 6 bar for 10 min to dry the membrane completely This step is necessary to eliminate traces of ethanol Note The ethanol in Buffer B5 inhibits enzymatic reactions and has to be removed completely before eluting DNA Finally release the vacuum Elute DNA Insert spacers Microtube rack into the NucleoVac 96 Vacuum Manifold s short sides Place a Rack of Tube Strips onto the spacer Close the vacuum mani fold and place the Column Holder A with the NucleoSpin Blood Binding Strips on top Dispense 50 200 pl Buffer BE directly to the bottom of each well Incubate for 5 min at room temperature Apply vacuum for elution 0 6 bar 1 min Dismantle the vacuum manifold and close Tube Strips with Cap Strips for storage Optional Preheat Buffer BE to 70 C to increase yield Reduction of atmospheric pressure MACHEREY NAGEL 02 2009 Rev 05 23 NucleoSpin 96 Blood 5 2 Nucle
7. the use of the Starter Set A is also required Centrifugation For centrifugation a microtiterplate centrifuge is required which is able to accommo date the NucleoSpin Blood Binding Strips Plate stacked on a Round or Square well Block and reaches accelerations of 5 600 6 000 x g bucket height 85 mm e g Hermle Z513 Qiagen Sigma 4 15c Jouan KR4i Kendro Heraeus Multifuge 3 3 R Highplate Beckman Coulter Allegra R25 For processing the 8 well strips the Starter Set C see ordering information containing Column Holders C NucleoSpin Dummy Strips MN Square well Blocks Rack of Tube Strips is required 1 5 Suitable vacuum manifolds The NucleoSpin 8 96 Blood kits can be used with the NucleoVac 96 Vacuum Manifold or other common vacuum devices For further details see list below Vacuum manifold Suitability Additional equipment NucleoVac 96 Yes Starter Set A for NucleoSpin 8 Blood Qiagen QlAvac 96 Yes MN Frame see ordering information Starter Set A for NucleoSpin 8 Blood Promega Vac Man 96 Yes NucleoSpin 96 Blood only 1 In general the QlAvac 96 is suitable for the use with the NucleoSpin Blood Binding Strips Plate Nevertheless it is recommended to use the MN Frame to adjust the proper height of the MN Wash Plate and Elution Plate in order to ensure best performance MN Wash Plate cannot be used 8 MACHEREY NAGEL 02 2009 Rev 05 Genomic DNA from Blood 2 2 1 P
8. 40475 4 sets Strips 740475 24 24 sets set consists of 1 Round well Block 12 Cap Strips MN Wash Plate 740479 4 740479 24 24 MN Square well Block 740476 4 740476 24 24 Starter Set A 740682 1 set for use of 8 well strips on the NucleoVac 96 and automation platforms Starter Set C 740684 1 set for use of 8 well strips under cen trifugation MN Frame 740680 1 for optimized handling of 96 well plates with vacuum manifold on BioRobot 9600 9604 and 3000 Qiagen MultiPROBE II PerkinElmer Biomek 2000 and FX Beckman Coulter NucleoVac 96 Vacuum Manifold 740681 1 NucleoVac Vacuum Regulator 740641 1 Self adhering PE Foil 740676 50 MACHEREY NAGEL 02 2009 Rev 05 33 Genomic DNA from Blood 6 3 Reference Vogelstein B and D Gillespie 1979 Preparative and analytical purification of DNA from agarose Proc Natl Acad Sci USA 76 615 619 6 4 Product use restriction warranty NucleoSpin 8 96 Blood Core kit components were developed designed distribut ed and sold FOR RESEARCH PURPOSES ONLY They are suitable FOR IN VITRO USES ONLY No claim or representation is intended for its use to identify any specific organism or for clinical use diagnostic prognostic therapeutic or blood banking It is rather the responsibility of the user to verify the use of the NucleoSpin 8 96 Blood Core kit for a specific application range as the performance characteristic of this kit has not been ver
9. AGEL laboratories using MACHEREY NAGEL products MACHEREY NAGEL does not warrant the correctness of any of those applications Please contact MACHEREY NAGEL Germany Tel 49 0 24 21 969 270 e mail TECH BIO Q mn net com Last updated 12 2006 Rev 02 Trademarks BioRobot is a registered trademark of the Qiagen GmbH Corporation Multifuge 3 3 R Highplate is a brand of Hereaus Instrument GmbH amp Co NucleoSpin is a registered trademark of MACHEREY NAGEL GmbH amp Co KG QlAvac 96 is a registered trademark of Qiagen GmbH Corporation Vac Man is a registered trademark of Promega Corporation All used names and denotations can be brands trademarks or registered labels of their respective owner also if they are not special denotation To mention products and brands is only a kind of information i e it does not offend against trademarks and brands and can not be seen as a kind of recommendation or assessment Regarding these products or services we can not grant any guarantees regarding selection efficiency or opera tion MACHEREY NAGEL 02 2009 Rev 05 35
10. Genomic DNA from Blood User Manual NucleoSpin 8 Blood NucleoSpin 96 Blood NucleoSpin 96 Blood Core Kit February 2009 Rev 05 MACHEREY NAGEL MN Genomic DNA from Blood Table of contents 1 Components 1 1 1 2 1 3 1 4 1 5 Kit contents Reagent to be supplied by user Accessories supplied by user NucleoSpin 96 Blood Core Kit Required hardware Suitable vacuum manifolds 2 Product description 2 1 2 2 2 3 2 4 The basic principle Kit specifications Elution procedure Automated processing on robotic platforms 3 Storage conditions and preparation of working solutions 4 Safety instructions risk and safety phrases 5 General procedure 5 1 5 2 5 3 5 4 5 5 NucleoSpin 8 Blood vacuum processing NucleoSpin 96 Blood vacuum processing NucleoSpin 8 96 Blood support protocol for centrifuge processing Support protocol for modified lysis of blood samples Support protocol for cultured animal or human cells 6 Appendix 6 1 6 2 6 3 6 4 Troubleshooting Ordering information Reference Product use restriction warranty 0o 0o dO h A o 12 13 14 16 17 21 24 27 28 29 30 30 32 34 34 MACHEREY NAGEL 02 2009 Rev 05 Genomic DNA from Blood 1 Components 1 1 Kit contents NucleoSpin 8 Blood 12 x 8 preps 740664 60 x 8 preps 740664 5 Lysis Buffer BQ1 40 ml 2 x 100 ml Wash Buffer B5 Concentrate 100 ml 5 x 100 ml Wa
11. Spin Blood Binding Strips Note Do not moisten the rims of the individual wells while dispensing the samples as this may lead to cross contamination 4 Overlay with Buffer B5 Overlay crude lysate on the NucleoSpin Blood Binding Strips slowly 50 ul s with 150 pl Buffer B5 5 Bind DNA to silica membrane Apply vacuum until all lysates have passed through the wells of the NucleoSpin9 Blood Binding Strips 0 2 bar 5 min Release the vacuum 6 Wash silica membrane Add 600 pl Buffer BW to each well of the NucleoSpin Blood Binding Strips Apply vacuum 0 2 bar 3 min until all buffer has passed through the wells of the NucleoSpin Blood Binding Strips Release the vacuum Add 900 ul Buffer Buffer B5 to each well of the NucleoSpin Blood Binding Strips Apply vacuum 0 2 bar 1 min until all buffer has passed through the wells of the NucleoSpin Blood Binding Strips Release the vacuum Reduction of atmospheric pressure 22 MACHEREY NAGEL 02 2009 Rev 05 NucleoSpin 8 Blood Add 900 yl Buffer Buffer B5 to each well of the NucleoSpin Blood Binding Strips Apply vacuum 0 2 bar 1 min until all buffer has passed through the wells of the NucleoSpin Blood Binding Strips Release the vacuum Remove MN Wash Plate After the final washing step close the valve release the vacuum and remove the Column Holder A with inserted NucleoSpin Blood Binding Strips from the vacu um manifold Put it on a
12. TE E ea fafa tater a ba a a ia LL LLL LI LLLILI TIL LLLA a a a a a La La el kul lul hel kur ker kur kur he hel let lal kel kul hel hel Gaf kel kel kul ker kul kez kur kul he l i hedl Tdi tt Tel T ha tar hev Jer te her her er ar raz fr har LI Li Figure 1 DNA isolation from human blood samples Genomic DNA from 200 yl of human blood 96 samples was isolated using NucleoSpin 96 Blood on a Tecan Genesis instrument Of each eluate 100 pl elution volume 20 ul were loaded and analyzed on a 0 7 agarose gel Highly reproducible yields of high quality genomic DNA were obtained 10 MACHEREY NAGEL 02 2009 Rev 05 Genomic DNA from Blood Figure 2 Figure 3 samples undiluted Fluorescence F1 no template control P p 10 20 30 40 50 Cycle number Analysis of purified DNA by real time PCR LightCycler Genomic DNA from 200 ul of blood 96 samples was isolated using NucleoSpin 96 Blood on a Tecan Genesis instrument 2 ul of eluate 100 ul elution volume were am plified with the LightCycler SYBR Green kit and 28S primers 0 5 uM All samples were amplified with homogenous crossing point values Amplification plots indicate the absence of inhibitors M 1 M ee ee ee C NE IE NE MM Mo o o o ol 8 Cross contamination analysis Genomic DNA was purified from 200 yl of blood using Nuc
13. erature to the Lysis Block Do not moisten the rims of the well C Add75 pl Buffer BQ1 to each sample pipette up and down 3 times and mix by shaking 15 min at room temperature Alternatively pipette up and down 10 times and incubate 15 min at room tem perature D Add 400 ul Buffer BQ1 ethanol mix 1 1 v v to each well of the Lysis Block mix at least 2 times and transfer lysate total volume 700 yl to the NucleoSpin Blood Binding Plate E Overlay crude lysate on the NucleoSpin Blood Binding Plate slowly 50 ul s with 150 pl Buffer B5 Wait for 1 min before applying vacuum for binding Proceed with DNA binding step 28 MACHEREY NAGEL 02 2009 Rev 05 Suppotr protocol for cultured cells 5 5 Support protocol for cultured animal or human cells Before starting the preparation Check if Wash Buffer B5 and Proteinase K were prepared according to sec tion 3 Seal unused wells of NucleoSpin Blood Binding Strips with Self adhering PE Foil see ordering information Harvest cells Harvest cells maximum starting amount 2 x 109 and pellet them in the lysis vessel by centrifugation 300 x g 4 min Remove supernatant and resuspend cell pellets in 200 pl PBS Lyse cells Add 25 ul Proteinase K and 200 pl Buffer BQ1 to each well and shake lysis vessel at least 10 min at room temperature Complete lysis is important for optimal yields Optional Add 10 ul RNase 25 mg ml not supplied with the k
14. he NucleoSpin 8 96 Blood kit is designed primarily for vacuum processing centrifuge processing is also possible NucleoSpin 8 Blood For processing under centrifugation the Starter Kit C and a suitable centrifuge are re quired see section 1 4 For handling of the 8 well strips and the column holders refer to the protocol of the Starter Kit C The use of NucleoSpin Blood Binding Strips in a Column Holder C allows the isolation of up to n x 8 samples n 1 to 6 Insert as many of the NucleoSpin Blood Binding Strips as required into the same positions of each one of the two reusable column hold ers and place column holders onto MN Square well Block see ordering information Label the column holders or 8 well strips for later identification Always use 2 Column Holders C containing identical numbers of NucleoSpin Blood Binding Strips for cen trifugation This avoids the need to balance the centrifuge and allows multiples of 16 samples to be processed in parallel We recommend inserting the NucleoSpin Blood Binding Strips around the center of the column holder Follow the standard protocol as described in section 5 1 The vacuum steps are substi tuted by centrifugation of the Column Holder C with the NucleoSpin 8 Blood Strips at 5 600 6 000 x g for 3 min Drying of the silica membrane is achieved by centrifugation for 10 min after the second Buffer B5 washing step A separate drying step is not required During all centrifugation
15. he NucleoSpin 8 Blood kit Lysis can be performed in any appropriate microtube or in suitable 96 well plates We recommend usage of the Lysis Block or Rack of Tube Strips with Cap Strips see ordering information 1 Lyse samples Dispense 25 ul Proteinase K and 200 ul blood equilibrated to room tempera ture to each lysis tube well Add 200 pl Buffer BQ1 to each lysis tube well mix 3 times by pipetting up and down and incubate samples at least 10 min at room temperature or Add 200 pl Buffer BQ1 to each tube well Mix 3 times by pipetting up and down and shake samples during incubation Recommended are 10 min at 1250 rpm Shake at room temperature Prepare the NucleoVac 96 Vacuum Manifold Place waste tray into vacuum manifold base Insert spacers labeled MTP Multi 96 plate notched side up and rest the MN Wash Plate on them Close the manifold with the manifold lid MACHEREY NAGEL 02 2009 Rev 05 21 NucleoSpin 8 Blood Insert desired number of NucleoSpin Blood Binding Strips in the Column Holder A Use NucleoSpin Dummy Strips to close unused openings in the col umn holder Place Column Holder A with inserted NucleoSpin Blood Binding Strips on top of the manifold 2 Adjust DNA binding conditions Add 200 pl 96 100 ethanol to each lysis tube well mix at least 3 times High speed pipetting 400 pl sec should be used for optimal mixing 3 Load samples Transfer the samples to the Nucleo
16. ified to a specific organism This MACHEREY NAGEL product is shipped with documentation stating specifica tions and other technical information MACHEREY NAGEL warrants to meet the stated specifications MACHEREY NAGEL S sole obligation and the customer s sole remedy is limited to replacement of products free of charge in the event products fail to perform as warranted Supplementary reference is made to the general business terms and conditions of MACHEREY NAGEL which are printed on the price list Please contact us if you wish an extra copy MACHEREY NAGEL does not warrant against damages or defects arising in shipping and handling transport insurance for customers excluded or out of accident or im proper or abnormal use of this product against defects in products or components not manufactured by MACHEREY NAGEL or against damages resulting from such non MACHEREY NAGEL components or products MACHEREY NAGEL makes no other warranty of any kind whatsoever and SPECIFICALLY DISCLAIMS AND EXCLUDES ALL OTHER WARRANTIES OF ANY KIND OR NATURE WHATSOEVER DIRECTLY OR INDIRECTLY EXPRESS OR IMPLIED INCLUDING WITHOUT LIMITATION AS TO THE SUITABILITY REPRODUCTIVITY DURABILITY FITNESS FOR A PARTICULAR PURPOSE OR USE MERCHANTABILITY CONDITION OR ANY OTHER MATTER WITH RESPECT TO MACHEREY NAGEL PRODUCTS In no event shall MACHEREY NAGEL be liable for claims for any other damages whether direct indirect incidental compensatory foreseeable
17. ilica membrane No drops should stick to the walls of the columns 30 MACHEREY NAGEL 02 2009 Rev 05 Genomic DNA from Blood Clogging of the NucleoSpin Blood Binding Strip Plate f blood samples are too old and clotting occurs clogging Clogging of the NucleoSpin Blood Binding Strip Plate may appear of binding Check for blockage of NucleoSpin Blood Binding Strip Plate strip plate visually or automatically and remove supernatant Increase time and strength for vacuum processing Whole blood can be stored for several weeks at 4 C Freeze samples at 20 C if blood should be stored for a longer periods Contamina RNA carryover tion of Add 10 ul 25 mg ml RNase A to the sample after the incuba genomic DNA tion of step 2 as recommended for working with fresh unfro with RNA zen cells Carryover of ethanol Suboptimal e Be sure to remove all traces of Buffer B5 after the final wash performance ing step Dry the NucleoSpin Blood Binding Strip Plate for at of DNA in least 10 min with maximum vacuum downstream experiments e Following the final wash step place NucleoSpin Blood Binding Strip Plate in an incubator for 10 min at 70 C to evaporate ethanol Vacuum manifold Vacuum pressure is not sufficient e Check if the vacuum manifold lid fits tightly on the manifold base if vacuum is turned on Make sure that pump works properly and that any in line fil ters are not blocked Buffers Buffe
18. inase Buffer to each vial to each bottle to each bottle NucleoSpin 96 Blood Core Kit 4 x 96 preps Cat No 740456 4 Wash Buffer B5 4 x 100 ml Concentrate Add 400 ml ethanol to each bottle Proteinase K 4 x 75 mg lyophilized Add 3 35 ml Proteinase Buffer to each bottle The kit of 24 x 96 preparations Cat No 740665 24 consists of 6 x Cat No 740665 4 MACHEREY NAGEL 02 2009 Rev 05 15 Genomic DNA from Blood 4 Safety instructions risk and safety phrases The following components of the NucleoSpin 8 96 Blood kits contain hazardous con tents Wear gloves and goggles and follow the safety instructions given in this section Hazard contents Component Hazard symbol Risk phrases Safety phrases BQ1 Guanidine x Xn Harmful if swallowed R 22 hydrochloride Irritating to eyes 36 38 and skin BW Guanidine x Xn Flammable Harmful R 10 22 S 7 16 25 hydrochloride by if swallowed 36 38 isopropanol Irritating to eyes lt 25 and skin Proteinase K Proteinase K x Xn Irritating to eyes R S 22 24 lyophilized Xi respiratory system 36 37 38 26 36 37 and skin May cause 42 sensitization by inhalation Risk phrases R 10 Flammable R 22 Harmful if swallowed R 36 37 38 Irritating to eyes respiratory system and skin R 36 38 Irritating to eyes and skin R 42 May cause sensitization by inhalation Safety phrases S7 S 16
19. it see ordering infor mation to each well after incubation if genomic DNA has to be free of RNA Proceed with step 2 Adjust binding conditions of the standard protocol MACHEREY NAGEL 02 2009 Rev 05 29 Genomic DNA from Blood 6 Appendix 6 1 Troubleshooting Problem Possible cause and suggestions Poor DNA quality or yield Low concentration of leukocytes in the whole blood sample Prepare buffy coat from the blood sample Incomplete cell lysis Sample not thoroughly mixed with Buffer BQ1 Proteinase K Use of a shaker is recommended for optimal results Proteinase K digestion not optimal Do not add Proteinase K directly to Buffer BQ1 Increase incubation time Incubate for at least 10 min at RT Reagents not applied or restored properly Reagents not properly restored Add the indicated volume of Proteinase Buffer PB to the Proteinase K vial and 96 10096 ethanol to Buffer B5 Concentrate and mix Kit storage Store aliquots of the reconstituted Proteinase K at 20 C Store other kit components at room temperature Storage at low temperatures may cause salt precipitation Keep bottles tightly closed in order to prevent evaporation or contamination Suboptimal elution Elution efficiencies decrease dramatically if elution is done with buffers with pH lt 7 0 Use slightly alkaline elution buffer like Buffer BE pH 8 5 Be sure that all of the elution buffer gets into contact with the s
20. it follow the standard protocol see sec tion 5 2 and 5 3 Recommended accessories for use of the NucleoSpin 96 Blood Core Kits are avail able from MACHEREY NAGEL see ordering information Protocol Suitable consumables not Remarka step supplied with the core kits Lysis 4x Lysis Block per 4x96 preps or 4x Round well Block with Cap Strips per 4x96 preps Round well Blocks and Tube Strips or can be closed with Cap Strips 4x Rack of Tube Strips with Cap Strips per 4x96 preps Binding of 4x MN Wash Plate per 4x96 MN Wash Plate minimizes the risk DNA to the preps of cross contamination vacuum membrane processing only 2x MN Square well Block For waste collection during centrifu gation reusable Elution 4x Rack of Tubes Strips with Cap Strips per 4x96 preps Round well Blocks and Tube Strips d can be closed with Cap Strips 4x Round well Block with Cap Strips per 4x96 preps MACHEREY NAGEL 02 2009 Rev 05 7 Genomic DNA from Blood 1 4 Required hardware Vacuum processing The NucleoSpin 8 96 Blood kits can be used manually with the NucleoVac 96 Vacuum Manifold see ordering information Alternatively other suitable vacuum mani folds may be used For processing the 8 well strips the Starter Set A see ordering information containing Column Holders A and NucleoSpin Dummy Strips is required For automation on labo ratory platforms with standard 96 well plate vacuum chambers
21. leoSpin 96 Blood on a Biomek 2000 Beckman Coulter instrument Blood samples and PBS buffer were ar rayed in a checkerboard pattern For PCR detection of genomic DNA 2 ul of eluate were amplified on iCycler BIORAD with Haemochromatosis Primer and Taq Polymerase Life Technologies 35 cycles were performed 48 samples were analyzed No cross contamination was detected samples containing 200 ul of blood control samples without blood next to a well containing blood MACHEREY NAGEL 02 2009 Rev 05 11 Genomic DNA from Blood 2 3 Elution procedure Recovery of gDNA from the membrane depends on the elution volume Elution volumes of 50 200 ul are possible with an optimum of 100 125 ul dispensed volume The pu rity is not effected by the elution volume See table for correlation between dispensed elution buffer volume and typical recoveries following the standard protocol Table 2 Recovery volumes in correlation to applied elution volumes Dispensed elution volume 40 ul 60 ul 80 ul 100 ul 120 pl Recovered volume Vacuum 25 yl 45 yl 65 ul 85 ul 105 ul Centrifuge 38 yl 58 ul 78 yl 98 ul 118 pl If highest yield is required prewarming of the elution buffer to 70 C will give about 10 15 higher yields by supporting DNA recovery from the membrane Concentration ng pl Figure 4 50 40 DNA recovered ug 30 20 25 50 75 100 125 150 175 200 Dispensed elution buffer pl
22. lica mem 0 2 bar Column Holder A with 5 min NucleoSpin Blood Binding Strips and MN Wash Plate Reduction of atmospheric pressure MACHEREY NAGEL 02 2009 Rev 05 17 NucleoSpin 8 Blood 6 Wash silica membrane 600 ul BW 0 2 bar 3 min 900 pI BS 0 2 bar 1 min Column Holder A with NucleoSpin Blood Binding 900 pl B5 Strips and MN Wash Plate 0 2 bar 1 min 7 Dry silica membrane Remove MN Wash Plate 0 6 bar 10 min 8 Elute DNA 50 200 ul BE Incubate 5 min 0 6 bar 1 min Column Holder A with NucleoSpin Blood Binding Strips on Rack of Tube Strips Reduction of atmospheric pressure 18 MACHEREY NAGEL 02 2009 Rev 05 NucleoSpin 96 Blood NucleoSpin 96 Blood vacuum processing For details on each step see section 5 2 Before starting the preparation e Check if Wash Buffer B5 and Proteinase K were prepared according to sec tion 3 1 Lyse samples 200 pl blood equilibrated to room tem perature 25 yl Proteinase K 200 yl BQ1 Mix 3 times Incubate at RT 10 min or Lysis Block Mix 3 times and shake at 1250 rpm at RT 10 min 2 Adjust DNA binding 200 yl ethanol conditions Mix at least 3 5 times Note High speed pipetting 400 l s should be used for optimized mixing if possible 3 Load samples Transfer samples to NucleoSpin Blood Binding Plate 4 Overlay with Buffer B5 150 ul B5 NucleoSpin Blood
23. m the vacuum manifold Reduction of atmospheric pressure MACHEREY NAGEL 02 2009 Rev 05 25 NucleoSpin 96 Blood Dry silica membrane Remove any residual washing buffer from the outlets of the NucleoSpin Blood Binding Plate If necessary tap the outlets onto a clean paper sheet supplied with the MN Wash Plate or soft tissue until no further drops come out Insert the NucleoSpin Blood Binding Plate into the lid and close the manifold Apply maximum vacuum at least 0 6 bar for 10 min to dry the membrane com pletely This step is necessary to eliminate traces of ethanol Note The ethanol in Buffer B5 inhibits enzymatic reactions and has to be removed completely before eluting DNA Finally release the vacuum Elute DNA Insert spacers Microtube rack into the NucleoVac Vacuum Manifold s short sides Place a Rack of Tube Strips onto the spacer Close the vacuum manifold and place the NucleoSpin Blood Binding Plate on top Dispense 50 200 pl Buffer BE onto the membrane Incubate for 5 min at room temperature Apply vacuum for elution 0 6 bar 1 min Dismantle the vacuum manifold and close Tube Strips with Cap Strips for storage Optional Preheat Buffer BE to 70 C to increase yield Reduction of atmospheric pressure 26 MACHEREY NAGEL 02 2009 Rev 05 Processing under centrifugation 5 3 NucleoSpin 8 96 Blood support protocol for centrifuge processing Although t
24. nto the automated procedure The MN Frame see ordering in formation can be used to position the MN Wash Plate inside the vacuum chamber Thorough cleaning of the vacuum chamber is recommended after each run to prevent forming of gDNA containing aerosols Visit MN on the internet at www mn net com or contact your local MACHEREY NAGEL distributor for technical support regarding hardware software setup instruc tions and selection of the protocol Several application notes of the NucleoSpin 8 96 Blood kit on various automation workstations can also be found at www mn net com at Bioanalyis Literature DNA yield ug DNA purity Azco Azso o be a a a a m u z z ar 0 0 8 16 24 32 40 48 45 64 72 80 88 96 0 8 16 24 32 40 48 45 64 72 80 88 96 Number of sample Number of sample Figure 5 Automated DNA isolation from human blood samples DNA was purified from 96 aliquots from the same blood using NucleoSpin 96 Blood on a Tecan Genesis 150 instrument vacuum processing The average yield is 6 76 ug 0 71 with a coefficient of variation CV of 10 The average purity is 1 97 0 02 CV 196 MACHEREY NAGEL 02 2009 Rev 05 13 Genomic DNA from Blood 3 Storage conditions and preparation of working solutions Attention Buffers BQ1 and BW contain guanidinium hydrochloride Wear gloves and goggles when handling them e All components of the NucleoSpin 8 96 Blood kits should be stored at room temperature for
25. o 740665 4 For preparation of working solutions and storage conditions see section 3 3 Elution Buffer BE 5 mM Tris HCl pH 8 5 For use with vacuum only 5 Sets of 1 rack 12 strips with 8 tubes each including Cap Strips MACHEREY NAGEL 02 2009 Rev 05 5 Genomic DNA from Blood 1 1 Kit contents continued NucleoSpin 96 Blood Core Kit 4 x 96 preps Cat No 740456 4 Lysis Buffer BQ1 125 ml Wash Buffer B5 Concentrate 4 x 100 ml Wash Buffer BW 2 x 300 ml Elution Buffer BE 125 ml Proteinase K lyophilized 4x 75 mg Proteinase Buffer PB 15 ml NucleoSpin Blood Binding 4 Plates red rings User Manual 1 Additional material required see section 1 3 1 2 Reagent to be supplied by user e 96 100 ethanol for preparation of working solutions see section 3 For preparation of working solutions and storage conditions see section 3 2 Elution Buffer BE 5 mM Tris HCl pH 8 5 6 MACHEREY NAGEL 02 2009 Rev 05 Genomic DNA from Blood 1 3 Accessories supplied by user NucleoSpin 96 Blood Core Kit The NucleoSpin 96 Blood Core Kit provides the buffers Proteinase K and NucleoSpin Blood Binding Plates only Accessory plates e g lysis plates elution plates are not provided with the core kits The user can individually select additional consumables from a variety of suitable accessory plates according to his requirements for highest flexibility For usage of NucleoSpin 96 Blood Core K
26. oSpin 8 96 Blood kits allows simultaneous processing of up to 96 samples typically within less than 70 minutes NucleoSpin 8 96 Blood kits can be processed completely at room tempera ture NucleoSpin 8 96 Blood can be processed by vacuum or centrifugation The kits allow for easy automation on common liquid handling instruments For more information about the automation process and the availability of ready to run scripts for certain platforms please refer to section 2 4 and contact your local distributor or MN directly The NucleoSpin 8 96 Blood kits allow for the purification of multiples of 8 NucleoSpin 8 Blood or 96 samples NucleoSpin 96 Blood Both kits are supplied with accessory plates for highest convenience The NucleoSpin 96 Blood Core Kit provides the buffers Proteinase K and NucleoSpin Blood Binding Plate only Accessory components e g lysis plates elution plates are not provided with the core kit but can be individually selected from a variety of suitable accessories see section 6 2 for further information This allows high est flexibility for the user MACHEREY NAGEL 02 2009 Rev 05 9 Genomic DNA from Blood Table 1 Kit specifications at a glance Parameters NucleoSpin 8 96 Blood Core Sample material Up to 200 ul Typical DNA yield 4 6 ug Elution volume 100 ul DNA binding capacity 20 ug Be oso 1 8 1 9 Preparation time 35 min for 6 strips or 1 plate Application data
27. oSpin 96 Blood vacuum processing For processing of NucleoSpin 96 Blood under vacuum the NucleoVac 96 Vacuum Manifold is required see ordering information Alternatively other suitable vacuum manifolds may be used Before starting the preparation prepare Buffer B5 and Proteinase K solution see sec tion 3 for details Before starting the preparation Check if Wash Buffer B5 and Proteinase K were prepared according to sec tion 3 1 Lyse samples Dispense 25 ul Proteinase K and 200 ul blood equilibrated to room tempera ture to each well of the Lysis Block Add 200 pl Buffer BQ1 to each well mix 3 times by pipetting up and down and incubate samples at least 10 min at room temperature or Add 200 ul Buffer BQ1 to each well Mix 3 times by pipetting up and down and shake samples during incubation Recommended are 10 min at 1250 rpm Shake at room temperature Prepare the NucleoVac 96 Vacuum Manifold Place waste tray into vacuum manifold base Insert spacers labeled MTP Multi 96 plate notched side up and rest the MN Wash Plate on them Close the manifold with the manifold lid Place a NucleoSpin Blood Binding Plate on top of the manifold 2 Adjust DNA binding conditions Add 200 pl 96 100 ethanol to each well of the Lysis Block mix at least 3 times High speed pipetting 400 pl s should be used for optimal mixing 24 MACHEREY NAGEL 02 2009 Rev 05 NucleoSpin 96 Blood Load samples Tran
28. r volumes are not enough Buffers are delivered in sufficient but limited amounts Calculate the needed buffer volumes and pour an additional amount of 10 into the reservoirs e Do not fill back unused buffer from reservoir to the flask to avoid contaminations Ask MACHEREY NAGEL technical service for larger buffer volumes MACHEREY NAGEL 02 2009 Rev 05 31 Genomic DNA from Blood Splattering of eluate e If eluting with vacuum be sure that the distance between the outlets of the NucleoSpin Blood Binding Strip Plate and the Cross Tube Strips is minimized contamination Sample transfer Be sure that no liquid drops out of the tips while moving the tips 6 2 Ordering information Product Cat No Pack of NucleoSpin 8 Blood 740644 12 x 8 preps NucleoSpin 8 Blood 740644 5 60 x 8 preps NucleoSpin 96 Blood 740665 1 1 x 96 preps NucleoSpin 96 Blood 740665 4 4 x 96 preps NucleoSpin 96 Blood 740665 24 24 x 96 preps NucleoSpin 96 Blood Core Kit 740456 4 4 x 96 preps Buffer BQ1 740923 1 11 Buffer B5 Concentrate 740921 100 100 ml Buffer BW 740922 500 500 ml Proteinase K 740506 100 mg RNase A 740505 100 mg Lysis Block 740484 4 32 MACHEREY NAGEL 02 2009 Rev 05 Genomic DNA from Blood Product Cat No Pack of Rack of Tube Strips 740477 4 sets with Cap Strips 740477 24 24 sets set consists of 1 Rack 12 Tube Strips with 8 tubes each and 12 Cap Strips Round well Block with Cap 7
29. roduct description The basic principle With the NucleoSpin 8 96 Blood method genomic DNA is prepared from whole blood buffy coat or cultured cells Lysis is achieved by incubation of whole blood in a lysis buffer containing chaotropic ions in the presence of Proteinase K at room temperature For optimal lysis a microplate shaker is recommended Appropriate conditions for bind ing of DNA to the silica membrane in the NucleoSpin Blood Binding Strips or Plate are created by addition of ethanol to the lysate The binding process is reversible and specific to nucleic acids Contaminations are removed by three wash steps with etha nolic buffers Pure genomic DNA is finally eluted under low ionic strength conditions in a slightly alkaline elution buffer 2 2 Kit specifications NucleoSpin 8 96 Blood kits are designed for the rapid small scale prepara tion of highly pure genomic DNA from whole animal or human blood serum plasma or other body fluids The obtained DNA can be used directly as tem plate for PCR blotting or any kind of enzymatic reactions The kits provide reagents and consumables for purification of up to 20 ug aver age 4 6 ug of pure genomic DNA from 200 ul whole blood with an A A ratio between 1 8 and 1 9 and a typical concentration of 20 60 ng ul Fresh and frozen blood and blood treated either with EDTA citrate or heparin can be used The procedure is optimized for a sample volume of 200 ul Using the Nucle
30. sfer the samples from the Lysis Block to the NucleoSpin Blood Binding Plate Note Do not moisten the rims of the individual wells while dispensing the sam ples as this might lead to cross contamination Overlay with Buffer B5 Overlay crude lysate on the NucleoSpin Blood Binding Plate slowly 50 ul s with 150 pl Buffer B5 Bind DNA to silica membrane Apply vacuum until all lysates have passed through the wells of the NucleoSpin Blood Binding Plate 0 2 bar 5 min Release the vacuum Wash silica membrane Add 600 pl Buffer BW to each well of the NucleoSpin Blood Binding Plate Apply vacuum 0 2 bar 3 min until all buffer has passed through the wells of the NucleoSpin Blood Binding Plate Release the vacuum Add 900 yl Buffer Buffer B5 to each well of the NucleoSpin Blood Binding Plate Apply vacuum 0 2 bar 1 min until all buffer has passed through the wells of the NucleoSpin Blood Binding Plate Release the vacuum Add 900 yl Buffer Buffer B5 to each well of the NucleoSpin Blood Binding Plate Apply vacuum 0 2 bar 1 min until all buffer has passed through the wells of the NucleoSpin Blood Binding Plate Release the vacuum Remove MN Wash Plate After the final washing step close the valve release the vacuum and remove the NucleoSpin Blood Binding Plate Put it on a clean paper towel to remove residual EtOH containing wash buffer Remove manifold lid MN Wash Plate and waste container fro
31. sh Buffer BW 150 ml 2x375ml Elution Buffer BE 50 ml 2x125ml Proteinase K lyophilized 75 mg 5x 75 mg Proteinase Buffer PB 3 6 ml 18 ml sper MN Wash Plates 1 5 Rack of Tube Strips 1 5 Cap Strips 12 60 Tubes 2 ml for Proteinase K 4 20 Tubes 15 ml for BioRobot 8 40 9604 User Manual Material supplied by user Suitable lysis tubes or plates e g Rack of Tube Strips with Cap Strips Cat No 740477 4 sets see ordering information 1 For preparation of working solutions and storage conditions see section 3 2 Elution Buffer BE 5 mM Tris HCl pH 8 5 3 For use with vacuum only Set of 1 rack 12 strips with 8 tubes each Cap Strips included 4 MACHEREY NAGEL 02 2009 Rev 05 Genomic DNA from Blood 1 1 Kit contents continued NucleoSpin 96 Blood 1 x 96 preps 4x96 preps 24x96 preps Cat No 740665 1 740665 4 740665 24 Lysis Buffer BQ1 40 ml 125 ml 6 x 125 ml Wash Buffer B5 Concentrate 100 ml 4x 100 ml 24 x 100 ml Wash Buffer BW 125 ml 2 x 300 ml 12 x 300 ml Elution Buffer BE 50 ml 125 ml 6 x 125 ml Proteinase K lyophilized 75 mg 4x75mg 24 x 75 mg Proteinase Buffer PB 3 6 ml 15 ml 6x15ml NucleoSpin Blood Binding Plates red rings 1 1 2 MN Wash Plates 1 4 24 Lysis Blocks 1 4 24 Rack of Tube Strips 1 4 24 Tubes 2 ml for Proteinase K 4 16 96 Mu 15 ml for BioRobot 8 32 192 User Manual 1 1 6 1 The kit for 24 x 96 preparations Cat No 740665 24 consists of 6 x Cat N
32. steps the Column Holder C with the NucleoSpin 8 Blood Strips should be placed on an MN Square well Block see ordering information to col lect the waste During the elution step the Column Holder C with the NucleoSpin 8 Blood Strips are placed on top of a Rack of Tube Strips NucleoSpin 96 Blood Follow the standard protocol as described in section 5 2 The vacuum steps are sub stituted by centrifugation of the NucleoSpin Blood Binding Plate at 5 600 6 000 x g for 3 min Drying of the silica membrane is achieved by centrifugation for 10 min after the second Buffer B5 washing step A separate drying step is not required During all centrifugation steps the NucleoSpin Blood Binding Plate should be placed on an MN Square well Block see ordering information to collect the waste During the elution step the NucleoSpin Blood Binding Plate is placed on top of a Rack of Tube Strips MACHEREY NAGEL 02 2009 Rev 05 27 Support protocol for modified lysis 5 4 Support protocol for modified lysis of blood samples This modified lysis procedure may be used to increase the yield on some liquid han dling instruments e g instruments with 4 channel pipetting system or if the recom mended mixing speed of 400 ul s for the addition of ethanol to adjust binding conditions can not be achieved A Pre dispense 25 yl of Proteinase K solution to each well of the Lysis Block B Transfer 200 pl blood equilibrated to room temp
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