NucleoSpin® Blood - MACHEREY
Contents
1. 1 Lyse blood sample Pipette up to 2 mL blood or body fluid sample equili brated to room temperature and 150 uL Proteinase K into a 15 mL tube not provided If processing buffy coat do not use more than 1 mL and add 2 mL blood PBS to adjust the volume to 2 mL 150 uL If cultured cells are used resuspend up to 2 x 107 cells in a Proteinase K final volume of 2 mL PBS Add 2 mL Buffer BQ1 if processing less than 2 mL blood add one volume of Buffer BQ1 to the samples and vortex 2 mL BQ1 the mixture vigorously for 10 s Note Vigorous mixing is important to obtain high yield and Mix purity of DNA In t mpl t 56 C for 15 min cubate samples at 56 C for 15 56 C Let the samples cool down to room temperature before 15 min proceeding with addition of ethanol The lysate should become brownish during incubation with Buffer BQ1 Increase incubation time with Proteinase K up to 20 min and vortex once or twice during incubation if processing older or clotted blood samples 18 MACHEREY NAGEL 06 2014 Rev 14 NucleoSpin Blood L Adjust DNA binding conditions Add 2 mL ethanol 96 100 if processing less than 2 mL blood add 1 volume of ethanol to each sample and mix by inverting the tube 10 times Note High local ethanol concentration must be avoided by immediate mixing after addition Be sure that the lysate has cooled down to room temperature before loading it onto the column Loading2. Genomic DNA from blood User manual NucleoSpin Blood June 2014 Rev 14 MACHEREY NAGEL www mn net com Genomic DNA from blood Protocol at a glance Rev 14 Mini miai oma Mini NucleoSpin NucleoSpin NucleoSpin NucleoSpin Blood Blood L Blood XL Blood QuickPure Lyse blood samples 200 uL blood 2 mL blood 10 mL blood 200 uL blood 25 uL Pro K 150 uL Pro K 500 uL Pro K 25 uL Pro K 200 uL B3 2 mL BQ1 10 mL BQ1 200 uL BQ1 Mix Mix Mix Mix 70 C 56 C 56 C 70 C 10 15 min 10 15 min 5 10 15 min 10 15 min Adjust DNA binding 210 uL ethanol 2 mL ethanol 10 mL ethanol 200 uL ethanol conditions Bind DNA Load all i Load 3 mL Load 15 mL Load all 11 000 x g 4 500 x g 4 000 x g 11 000 x g 5 1 min 5 3 min 3 min 5 1 min Load 3 mL Load 15 mL of residue of residue es 4 500 x g 4 000 x g 5 min 3 min Wash silica membrane 500 uL BW 3 2 mL BQ2 7 5 mL BQ2 350 uL BQ2 600 uL B5 A 2 mL BQ2 7 5 mL BQ2 1 wash 11 000 x g 4 500 x g 4 000 x g 11 000 x g 5 1 min 2 min 5 2 min 3 min 2 4 wash es 11 000 x g es 4 500 x g es 4 000 x g 1 min 10 min 10 min a Dry silica Drying is performed Drying is performed Drying is performed membrane 11 000 x g during centrifuga during centrifuga during centrifuga 1 min tion of the last tion of the last tion of the last washing step washing step washing step Elute 79 100 uL BE 200 uL BE 5
3. BQ2 before eluting the DNA If the level of B5 BQ2 after the second wash has reached the column outlet for any reason discard flow through place the column back into the Collection Tube and centrifuge again MACHEREY NAGEL 06 2014 Rev 14 27 Genomic DNA from blood Suboptimal performance of genomic DNA in enzymatic reactions continued Contamination of DNA with inhibitory substances If DNA has been eluted with Tris EDTA buffer TE make sure that EDTA does not interfere with downstream applications or repurify DNA and elute in Buffer BE If preparing DNA from older or clotted blood samples extend Proteinase K incubation to 30 min and vortex once or twice during this step If the Azgo Asgo ratio of the eluate is below 1 6 repeat the purification procedure For NucleoSpin Blood Add 1 volume of Buffer B3 plus 1 volume ethanol to the eluate load on NucleoSpin Blood Column and proceed with step 3 of the corresponding protocol For NucleoSpin Blood QuickPure Add 1 volume of Buffer BQ1 plus 1 volume ethanol to the eluate load on NucleoSpin Blood QuickPure Column and proceed with step 3 of the corresponding protocol For NucleoSpin Blood L XL Add 1 volume of Buffer BQ1 plus 1 volume ethanol to the eluate load on NucleoSpin Blood L XL Column and proceed with step 3 of the corresponding protocol 28 MACHEREY NAGEL 06 2014 Rev 14 Genomic DNA from blood 6 2 Ordering info
4. NAGEL 06 2014 Rev 14 Genomic DNA from blood NucleoSpin Blood 10 preps 50 preps 250 preps REF 740951 10 740951 50 740951 250 Wash Buffer B5 6 mL 12 mL 50 mL Concentrate Add 24 mL ethanol Add 48 mL ethanol Add 200 mL ethanol Proteinase K 6 mg 30 mg 2x75mg Add 260 uL Add 1 35 mL Add 3 35 mL Proteinase Buffer Proteinase Buffer Proteinase Buffer to each vial NucleoSpin NucleoSpin NucleoSpin Blood L Blood XL Blood XL 20 preps 10 preps 50 preps REF 740954 20 740950 10 740950 50 Wash Buffer BQ2 20 mL 50 mL 4x50 mL Concentrate Add 80 mL ethanol Add 200 mL Add 200 mL ethanol ethanol to each bottle Proteinase K 60 mg 126 mg 5 x 126 mg Add 3 15 mL Add 5 75 mL Add 5 75 mL Proteinase Buffer Proteinase Buffer Proteinase Buffer to each vial NucleoSpin Blood QuickPure REF Wash Buffer BQ2 Concentrate Proteinase K 10 preps 50 preps 250 preps 740569 10 740569 50 740569 250 7mL 7mL 2x20 mL Add 80 mL ethanol to each bottle Add 28 mL ethanol Add 28 mL ethanol 6 mg 30 mg 2x 75mg Add 260 uL Add 1 35 mL Add 3 35 mL Proteinase Buffer Proteinase Buffer Proteinase Buffer to each vial MACHEREY NAGEL 06 2014 Rev 14 13 Genomic DNA from blood 4 Safety instructions The following components of the NucleoSpin Blood kits contain hazardous contents Wear gloves and goggles and follow the safety instructions given in this section GHS classification Only harmful features do no
5. and up to 200 uL blood body fluid sample or buffy coat from 1 mL blood equilibrated to room temperature into 1 5 mL microcentrifuge tubes not provided 200 uL blood For sample volumes less than 200 uL add PBS to adjust the volume to 200 uL If purifying DNA viruses we recommend 25 pL starting with 200 uL serum or plasma If cultured cells are Protei K used resuspend up to 5 x 10 cells in a final volume of 200 uL FOteInase PBS 200 pL B3 Add 200 pL Buffer B3 to the samples and vortex the i mixture vigorously 10 20 s Mix Note Vigorous mixing is important to obtain high yield and 70 C purity of DNA 10 15 min Incubate samples at 70 C for 10 15 min The lysate should become brownish during incubation with Buffer B3 Increase incubation time with Proteinase K up to 30 min and vortex once or twice vigorously during incubation if processing older or clotted blood samples 2 Adjust DNA binding conditions 210 uL ethanol Add 210 uL ethanol 96 100 to each sample and vortex again Mix 16 MACHEREY NAGEL 06 2014 Rev 14 NucleoSpin Blood Bind DNA For each preparation take one NucleoSpin Blood Column placed in a Collection Tube and load the sample Centrifuge 1 min at 11 000 x g If the samples are not drawn through the matrix completely repeat the centrifugation at higher g force lt 15 000 x g Discard Collection Tube with flow through Wash silica membrane Place the NucleoSpin Blood C
6. consequential or special 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 the product and warranty No other statements or representations written or oral by MACHEREY NAGEL s employees agent or representatives except written statements 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 NAGEL laboratories using MACHEREY NAGEL products MACHEREY NAGEL does not
7. fields of application Application on the human body is STRICTLY FORBIDDEN The respective user is liable for any and all damages resulting from such application DNA RNA PROTEIN purification products of MACHEREY NAGEL are suitable for N VITRO USES ONLY ONLY MACHEREY NAGEL products specially labeled as IVD are also suitable for IN VITRO diagnostic use Please pay attention to the package of the product IN V TRO diagnostic products are expressly marked as IVD on the packaging IF THERE IS NO IVD SIGN THE PRODUCT SHALL NOT BE SUITABLE FOR IN VITRO DIAGNOSTIC USE ALL OTHER PRODUCTS NOT LABELED AS IVD ARE NOT SUITED FOR ANY CLINICAL USE INCLUDING BUT NOT LIMITED TO DIAGNOSTIC THERAPEUTIC AND OR PROGNOSTIC USE No claim or representations is intended for its use to identify any specific organism or for clinical use included but not limited to diagnostic prognostic therapeutic or blood banking It is rather in the responsibility of the user or in any case of resale of the products in the responsibility of the reseller to inspect and assure the use of the DNA RNA protein purification products of MACHEREY NAGEL for a well defined and specific application MACHEREY NAGEL shall only be responsible for the product specifications and the performance range of MN products according to the specifications of in house quality control product documentation and marketing material This MACHEREY NAGEL product is shipped with documentat
8. obtained DNA is ready to use for subsequent reactions like PCR Southern blotting or any kind of enzymatic reactions MACHEREY NAGEL 06 2014 Rev 14 9 Genomic DNA from blood Table 1 Kit specifications at a glance Parameter Blood Blood L Blood XL Bogd QuickPure Up to 200 uL Upto2mL Upto10mL Upto 200 uL 5x10 cells 2x10 cells 1x108 cells 5x 10 cells Sample material Typical yield 4 6 ug 40 60 ug 200 300 ug 4 6 ug Elution volume 60 200 uL 120 200 uL 600 2000 uL 30 50 uL Binding capacity 60 ug 250 ug 700 ug 50 ug Preparation time 30 min prep 1 h prep 1 h prep lt 10 min prep Format Mini Midi Maxi Mini spin column spin column spin column spin column 2 3 Storage of blood samples For the isolation of genomic DNA from blood treated with anticoagulants heparin citrate or EDTA using a NucleoSpin Blood kit the blood samples can be stored at room temperature 4 C or frozen Blood samples stored at room temperature or 4 C for up to several days or weeks respectively will still allow DNA isolation However DNA yield and quality will slowly decrease due to prolonged storage of blood samples under these conditions Blood stored frozen for years is well suited for DNA isolation Highest yields and quality of DNA are obtained from fresh blood 2 4 Elution procedures It is possible to adapt elution method and volume of elution buffer to the subsequent application of interest In addition to th
9. 00 uL highly 70 C 70 C 2000 uL BE pure DNA 70 C RT RT RT 1 min v 2 min 2 min 11 000 x g 4 500 x g 4 000 x g 11 000 x g 5 1 min 5 2 min 2 min 5 1 min MACHEREY NAGEL GmbH amp Co KG Neumann Neander Str 6 8 52355 D ren Germany Tel 49 24 21 969 270 Fax 49 24 21 969 199 tech bio mn net com www mn net com Genomic DNA from blood Table of contents 1 Components 4 1 1 Kit contents 4 1 2 Reagents consumables and equipment to be supplied by user 8 1 3 About this user manual 8 2 Product description 9 2 1 The basic principle 9 2 2 Kit specifications 9 2 3 Storage of blood samples 10 2 4 Elution procedures 10 3 Storage conditions and preparation of working solutions 12 4 Safety instructions 14 5 Protocols for DNA purification from whole blood 16 5 1 Genomic DNA purification with NucleoSpin Blood 16 5 2 Genomic DNA purification with NucleoSpin Blood L 18 5 3 Genomic DNA purification with NucleoSpin Blood XL 21 5 4 Genomic DNA purification with NucleoSpin Blood QuickPure 24 6 Appendix 26 6 1 Troubleshooting 26 6 2 Ordering information 29 6 3 Reference 29 6 4 Product use restriction warranty 30 MACHEREY NAGEL 06 2014 Rev 14 3 Genomic DNA from blood 1 Components 1 1 Kit contents NucleoSpin Blood 10 preps 50 preps 250 preps REF 740951 10 740951 50 740951 250 Buffer B3 10 mL 15 mL 60 mL Wash Buffer BW 6 mL 30 mL 150 mL Wash Buffer BS 6mL 12 mL 50
10. Add 7 5 mL Buffer BQ2 to the NucleoSpin Blood XL 4 000 x g Column Centrifuge 2 min at 4 000 x g 2 min It is not necessary to discard the flow through after the first washing step Add 7 5 mL Buffer BQ2 Centrifuge 10 min at 4 000 x g S 47 5mLBQ2 Remove the column carefully from the rotor to avoid that flow through gets in contact with the column outlet 4 000 x g By prolonged centrifugation during this second washing step 10min residual ethanolic Buffer BQ2 is removed from the silica membrane of the NucleoSpin Blood XL Column 5 Dry silica membrane The drying of the NucleoSpin Blood XL Column is performed by prolonged centrifugation time 10 min in the 2 wash step 6 Elute highly pure DNA 1000 uL BE r 70 C Insert the column into a new Collection Tube 50 mL and apply 1000 uL of preheated Buffer BE 70 C directly RT to the center of the silica membrane Incubate at room 2 min temperature for 2 min Centrifuge at 4 000 x g for 2 min For alternative elution procedures see section 2 4 4 000 x g S 2 min MACHEREY NAGEL 06 2014 Rev 14 23 NucleoSpin Blood QuickPure 5 4 Genomic DNA purification with NucleoSpin Blood QuickPure Before starting the preparation Check if Buffer BQ2 and Proteinase K were prepared according to section 3 Set an incubator or water bath to 70 C Preheat Elution Buffer BE to 70 C Lyse blood sample Pipette 25 uL Proteinase K and up to 200 uL blo
11. DNA from blood 3 Storage conditions and preparation of working solutions Attention Buffers B3 BQ1 and BW contain chaotropic salt Wear gloves and goggles CAUTION Buffer B3 BQ1 and BW contain guanidine hydrochloride which can form highly reactive compounds when combined with bleach sodium hypochlorite DO NOT add bleach or acidic solutions directly to the sample preparation waste All kit components can be stored at room temperature 18 25 C and are stable up to one year During storage especially at low temperatures a white precipitate may form in Buffer T1 B3 or BQ1 Such precipitates can be easily dissolved by incubating the bottle at 70 C before use Before starting any NucleoSpin Blood protocol prepare the following Wash Buffer B5 NucleoSpin Blood Add the indicated volume of ethanol 96 100 to Wash Buffer B5 Concentrate Mark the label of the bottle to indicate that ethanol was added Store Wash Buffer B5 at room temperature 18 25 C for up to one year Wash Buffer BQ2 NucleoSpin Blood L XL QuickPure Add the indicated volume of ethanol 96 100 to Wash Buffer BQ2 Concentrate Mark the label of the bottle to indicate that ethanol was added Store Wash Buffer BQ2 at room temperature 18 25 C for up to one year Proteinase K Add the indicated volume of Proteinase Buffer PB to dissolve lyophilized Proteinase K Proteinase K solution is stable at 20 C for up to 6 months 12 MACHEREY
12. ation take one NucleoSpin Blood XL Column placed in a Collection Tube and load 15 mL of lysate Do not moisten the rim of the column Close the tubes with screw caps and centrifuge 3 min at 4 000 x g Discard flow through Discarding the flow through may be omitted Be careful after the second loading step during removal of the tube from the centrifuge and the removal of the column from the tube keep tube with column upright to avoid contact of flow through with the column outlet Usually the lysate will start to flow through the column even before centrifugation This will not adversely affect DNA yield or purity Keep NucleoSpin Blood XL Column in an upright position as liquid may pass through the ventilation slots on the rim of the column even if the caps are closed Load 15 mL of the remaining lysate to the respective NucleoSpin Blood XL Column Again avoid moistening the rim Centrifuge 3 min at 4 000 x g Discard the flow through and place the column back into the Collection Tube Remove the Collection Tube with the column carefully from the rotor and avoid that the flow through comes in contact with the column outlet If you process lt 5 mL blood no loading of remaining lysate is necessary amp 10 mL ethanol Mix Load 15 mL 4 000xg 3 min Load residue 4 000xg 3 min 22 MACHEREY NAGEL 06 2014 Rev 14 NucleoSpin Blood XL 4 Wash silica membrane 1 wash 7 5 mL BQ2
13. bei Einatmen Allergie asthmaartige Symptome oder Atembeschwerden verursachen H 335 May cause respiratory irritation Kann die Atemwege reizen 14 MACHEREY NAGEL 06 2014 Rev 14 Genomic DNA from blood Precaution phrases P 210 P 233 P 261 P 280 P 301 312 P 302 352 P 304 340 P 305 351 338 P 312 P 330 P 332 313 P 337 313 P 342 311 P 403 233 P 403 235 Keep away from heat hot surfaces sparks open flames and other ignition sources No smoking Von Hitze hei en Oberfl chen Funken offenen Flammen sowie anderen Z ndquellenarten fernhalten Nicht rauchen Keep container tightly closed Beh lter dicht verschlossen halten Avoid breathing dust Einatmen von Staub vermeiden Wear protective gloves eye protection Schutzhandschuhe Augenschutz tragen IF SWALLOWED Call a POISON CENTER doctor if you feel unwell BEI VERSCHLUCKEN Bei Unwohlsein GIFTINFORMATIONSZENTRUM Arzt anrufen IF ON SKIN Wash with plenty of water BEI KONTAKT MIT DER HAUT Mit viel Wasser und Seife waschen IF INHALED Remove victim to fresh air and keep at rest in a position com fortable for breathing BEI EINATMEN An die frische Luft bringen und in einer Position ruhigstellen die das Atmen erleichtert IF IN EYES Rinse continuously with water for several minutes Remove contact lenses if present and easy to do continue rinsing BEI KONTAKT MIT DEN AUGEN Einige Minuten lang b
14. d 350 uL Buffer BQ2 Centrifuge 3 min at 11 000 x g Discard Collection Tube with flow through Optional Place the NucleoSpin Blood QuickPure Column into a new Collection Tube 2 mL not provided and add 200 uL Buffer BQ2 Centrifuge 1 min at 11 000 x g Discard flow through and Collection Tube and proceed to step 6 This additional washing step is only recommended if the DNA is intended for use as a template in especially critical PCRs In the vast majority of cases you can save time by this step Dry silica membrane The drying of the NucleoSpin Blood QuickPure Column is performed by the 3 min centrifugation in step 4 Elute highly pure DNA Place the NucleoSpin Blood QuickPure Column in a 1 5 mL microcentrifuge tube not provided and add 50 pL prewarmed Buffer BE 70 C Dispense buffer directly onto the silica membrane Incubate at room temperature for 1 min Centrifuge 1 min at 11 000 x g For alternative elution procedures see section 2 4 350 pL BQ2 11 000 x g 3 min 50 pL BE 70 C RT 1 min 11 000 x g 1 min MACHEREY NAGEL 06 2014 Rev 14 25 Genomic DNA from blood 6 Appendix 6 1 Troubleshooting Problem Possible cause and suggestions Low concentration of leukocytes in sample Prepare buffy coat from the blood sample Centrifuge whole blood at room temperature 3 300 x g 10 min Three different layers will be visible after centrifugation Leukocytes ar
15. d QuickPure kit contaminations are removed by a single wash step Pure genomic DNA is finally eluted under low ionic strength conditions in a slightly alkaline elution buffer 2 2 Kit specifications NucleoSpin Blood kits are designed for the rapid isolation of highly pure genomic DNA from whole blood serum plasma or other body fluids It is also possible to purify viral DNA e g HBV from blood samples As viral DNA co purifies with cellular DNA we recommend using cell free samples serum or plasma to prepare pure viral DNA The NucleoSpin Blood QuickPure kit is designed for ultra fast small scale purification of highly pure genomic DNA from whole blood serum plasma or other body fluids The number of washing and drying steps is reduced from 3 to 1 Therefore the hands on time is less than 10 min DNA can be purified successfully from blood samples treated with EDTA citrate or heparin If leukocyte rich materials like buffy coat are used apply smaller volumes and dilute the samples with sterile PBS dissolve 8 g NaCl 0 2 g KCI 1 44 g Na HPO and 0 24 g KH PO in 800 mL H O Adjust pH to 7 4 with HCI Add H 0 to 1 liter The kits allow purification of highly pure genomic DNA with an Axgo Azgo ratio between 1 60 and 1 90 and a typical concentration of 40 60 ng per uL for the NucleoSpin Blood kit 80 120 ng per uL for the NucleoSpin Blood QuickPure kit and 200 300 ng per uL for the NucleoSpin Blood L XL kits The
16. e concentrated in the intermediate layer buffy coat Incomplete cell lysis Sample not thoroughly mixed with lysis buffer Proteinase K The mixture has to be vortexed vigorously immediately after addition of lysis buffer Proteinase K digestion is not optimal Never add Proteinase K directly to lysis buffer Incubate for 15 20 min at 70 C 56 C No or poor DNA yield Reagents not applied properly Prepare buffers and Proteinase K solution according to instruc tions section 3 Add ethanol to lysates before loading them on columns Suboptimal elution of DNA from the column Preheat Buffer BE to 70 C before elution Apply Buffer BE directly onto the center of the silica membrane Elution efficiencies decrease dramatically if elution is performed with buffers of pH lt 7 0 Use slightly alkaline elution buffer like Buffer BE pH 8 5 e Mix vigorously once during the 70 C 56 C incubation step especially when working with older or clotted blood samples Reagents not applied properly Prepare buffers and Proteinase K solution according to instructions section 3 Add ethanol to lysates and mix before loading them on columns Poor DNA quality Incomplete cell lysis Sample not thoroughly mixed with lysis buffer Proteinase K The mixture has to be vortexed vigorously immediately after addition of lysis buffer 26 MACHEREY NAGEL 06 2014 Rev 14 Genomic DNA from blood Poor DNA quality continued S
17. e 10 min at 4 500 x g Remove the column carefully from the rotor in order to e gt 2 mL BQ2 avoid that the flow through comes in contact with the column outlet 4 500 x g 10 min By prolonged centrifugation during this second washing step residual ethanolic washing Buffer BQ2 is removed from the silica membrane of the NucleoSpin Blood L Column 5 Dry silica membrane The drying of the NucleoSpin Blood L Column is performed by prolonged centrifugation time 10 min in the 2 wash step 6 Elute highly pure DNA 200 uL BE F 70 C Insert the column into a new Collection Tube 15 mL and apply 200 pL preheated Buffer BE 70 C directly RT to the center of the silica membrane Incubate at room imit temperature for 2 min Centrifuge at 4 500 x g for 2 min For alternative elution procedures see section 2 4 e gt 4 500 x g 2 min 20 MACHEREY NAGEL 06 2014 Rev 14 NucleoSpin Blood XL 5 3 Genomic DNA purification with NucleoSpin Blood XL Before starting the preparation Check if Buffer BQ2 and Proteinase K were prepared according to section 3 Set an incubator or water bath to 56 C Preheat Elution Buffer BE to 70 C For centrifugation a centrifuge with a swing out rotor and appropriate buckets capable of reaching 4 000 4 500 x gis required 1 Lyse blood sample Pipette up to 10 mL blood or body fluid sample equili brated to room temperature and 500 uL Proteinase K into a 50 mL tube not provided If y
18. e standard method recovery rate about 70 90 there are several modifications possible Use elution buffer preheated to 70 C for one of the following procedures High yield Perform two elution steps with the volume indicated in the individual protocol About 90 100 of bound nucleic acid can be eluted High concentration Perform one elution step with 60 of the volume indicated in the individual protocol Concentration of DNA will be higher than with standard elution NucleoSpin Blood ca 130 NucleoSpin Blood QuickPure ca 150 NucleoSpin Blood L ca 140 NucleoSpin Blood XL ca 115 Maximal yield of bound nucleic acid is about 80 10 MACHEREY NAGEL 06 2014 Rev 14 Genomic DNA from blood High yield and high concentration Apply half the volume of elution buffer as indicated in the individual protocol incubate for 3 min and centrifuge Apply a second aliquot of elution buffer incubate and centrifuge again Thus about 85 100 of bound nucleic acid is eluted in the standard elution volume at a high concentration Convenient elution For convenience elution buffer of ambient temperature may be used This will result in a lower yield approximately 20 compared to elution with preheated elution buffer Elution may also be performed with Tris EDTA buffer TE of pH equal or higher than 8 This will increase DNA stability especially during long term and or multi use storage at 4 C or ambient tempe
19. ehutsam mit Wasser sp len Vorhandene Kontaktlinsen nach M glichkeit entfernen Weiter sp len Call a POISON CENTER doctor if you feel unwell Bei Unwohlsein GIFTINFORMATIONSZENTRUM Arzt anrufen Rinse mouth Mund aussp len If skin irritation occurs Get medical advice attention Bei Hautreizung Arztlichen Rat einholen rztliche Hilfe hinzuziehen Get medical advice attention Bei anhaltende Augenreizung Arztlichen Rat einholen rztliche Hilfe hinzuziehen If experiencing respiratory symptoms Call a POISON CENTERF doctor Bei Symptomen der Atemwege GIFTINFORMATIONSZENTRUM Arzt anrufen Store in a well ventilated place Keep container tightly closed Beh lter dicht verschlossen an einem gut bel fteten Ort aufbewahren Store in a well ventilated place Keep cool K hl an einem gut bel fteten Ort aufbewahren For further information please see Material Safety Data Sheets www mn net com Weiterf hrende Informationen finden Sie in den Sicherheitsdatenbl ttern www mn net com MACHEREY NAGEL 06 2014 Rev 14 15 NucleoSpin Blood 5 5 1 Protocols for DNA purification from whole blood Genomic DNA purification with NucleoSpin Blood Before starting the preparation Check if Buffer B5 and Proteinase K were prepared according to section 3 Set an incubator or water bath to 70 C Preheat Elution Buffer BE to 70 C Lyse blood sample Pipette 25 uL Proteinase K
20. ion stating specifications 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 to get an extra copy There is no warranty for and MACHEREY NAGEL is not liable for damages or defects arising in shipping and handling transport insurance for customers excluded or 30 MACHEREY NAGEL 06 2014 Rev 14 Genomic DNA from blood out of accident or improper or abnormal use of this product defects in products or components not manufactured by MACHEREY NAGEL or 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
21. mL Concentrate Elution Buffer BE 13 mL 13 mL 60 mL Proteinase K lyophilized 6 mg 30 mg 2x75 mg Proteinase Buffer PB 1 8 mL 1 8 mL 8 mL NucleoSpin Blood Columns red rings plus 10 50 250 Collection Tubes Collection Tubes 2 mL 20 100 500 User manual 1 1 1 For preparation of working solutions and storage conditions see section 3 Composition of Elution Buffer BE 5 mM Tris HCl pH 8 5 4 MACHEREY NAGEL 06 2014 Rev 14 Genomic DNA from blood 1 1 Kit contents continued NucleoSpin Blood L 20 preps REF 740954 20 Lysis Buffer BQ1 45 mL Wash Buffer BQ2 Concentrate 20 mL Elution Buffer BE 13 mL Proteinase K lyophilized 60 mg Proteinase Buffer PB 8 mL NucleoSpin Blood L Columns 20 plus Collection Tubes Collection Tubes 15 mL 20 User manual 1 For preparation of working solutions and storage conditions see section 3 Composition of Elution Buffer BE 5 mM Tris HCl pH 8 5 MACHEREY NAGEL 06 2014 Rev 14 5 Genomic DNA from blood 1 1 Kit contents continued NucleoSpin Blood XL 10 preps 50 preps REF 740950 10 740950 50 Lysis Buffer BQ1 125 mL 3x200 mL Wash Buffer BQ2 Concentrate 50 mL 4x50 mL Elution Buffer BE 30 mL 125 mL Proteinase K lyophilized 126 mg 5 x 126 mg Proteinase Buffer PB 8 mL 35 mL NucleoSpin Blood XL Columns 10 50 plus Collection Tubes Collection Tubes 50 mL 10 50 User manual 1 1 For preparation of working sol
22. od buffy coat or body fluid sample equilibrated to room temperature into 1 5mL microcentrifuge tubes not provided For sample volumes less than 200 uL add PBS to adjust the volume to 200 uL If cultured cells are used resuspend up to 5 x 10 cells in a final volume of 200 uL PBS Add 200 uL Lysis Buffer BQ1 to the samples and vortex the mixture vigorously 10 20 s Note Vigorous mixing is important to obtain high yield and purity of DNA Incubate samples at 70 C for 10 15 min The lysate should become brownish during incubation with Buffer BQ1 Increase incubation time with Proteinase K up to 30 min and vortex once or twice vigorously during incubation if processing older or clotted blood samples 200 uL blood 25 pL Proteinase K y 200 uL BQ1 Mix 70 C 10 15 min 2 Adjust DNA binding conditions 200 uL ethanol Add 200 uL ethanol 96 100 to each sample and vortex again Mix 3 Bind DNA Apply the samples to the NucleoSpin Blood QuickPure Load lysate Columns placed in a Collection Tube and centrifuge 1 min at 11 000 x g If the samples are not drawn through the matrix completely repeat the centrifugation at higher 11 000 x g g force up to 15 000 x g Discard Collection Tube with 1 min flow through 24 MACHEREY NAGEL 06 2014 Rev 14 NucleoSpin Blood QuickPure Wash silica membrane Place the NucleoSpin Blood QuickPure Column into a new Collection Tube 2 mL and ad
23. of hot lysate may lead to diminished yields Bind DNA For each preparation take one NucleoSpin Blood L Column placed in a Collection Tube and load 3 mL of lysate Do not moisten the rims of the columns Close the tubes with screw caps and centrifuge 3 min at 4 500 x g Usually the lysate will start to flow through the columns even before centrifugation This will not adversely affect DNA yield or purity Keep NucleoSpin Blood L Column in an upright position as liquid may pass through the ventilation slots on the rim of the column even if the caps are closed Load all of the remaining lysate in a second step to the respective NucleoSpin Blood L Column avoiding moist ening the rim Centrifuge 5 min at 4 500 x g Discard the flow through and place the column back into the Collection Tube Remove the Collection Tube with the column carefully from the rotor to avoid that the flow through comes in contact with the column outlet Be sure to wipe off any spilled lysate from the Collection Tube before placing the column back 2 mL ethanol Mix Load 3 mL 4 500 x g v 3 min Load residue 4 500 x g 5 min MACHEREY NAGEL 06 2014 Rev 14 19 NucleoSpin Blood L 4 Wash silica membrane 1 wash ne Add 2 mL Buffer BQ2 Centrifuge 2 min at 4 500 x g anaes g min It is not necessary to discard the flow through after the first washing step ET y Add 2 mL Buffer BQ2 Centrifug
24. olumn into a new Collection Tube 2 mL and add 500 uL Buffer BW Centrifuge 1 min at 11 000 x g Discard Collection Tube with flow through Place the NucleoSpin Blood Column into a new Collection Tube 2 mL and add 600 uL Buffer B5 Centrifuge 1 min at 11 000 x g Discard flow through and reuse Collection Tube Dry silica membrane Place the NucleoSpin Blood Column back into the Collection Tube and centrifuge 1 min at 11 000 x g Residual ethanol is removed during this step Elute highly pure DNA Place the NucleoSpin Blood Column in a 1 5 mL microcentrifuge tube not provided and add 100 uL preheated Buffer BE 70 C Dispense buffer directly onto the silica membrane Incubate at room temperature for 1 min Centrifuge 1 min at 11 000 x g For alternative elution procedures see section 2 4 Load lysate 11 000 x g 1 min 500 pL BW 11 000 x g 1 min 600 uLB5 11 000 x g 1 min 11 000 x g 1 min 100 pL BE 70 C RT 1 min 11 000 x g 1 min MACHEREY NAGEL 06 2014 Rev 14 17 NucleoSpin Blood L 5 2 Genomic DNA purification with NucleoSpin Blood L Before starting the preparation Check if Buffer BQ2 and Proteinase K were prepared according to section 3 Set an incubator or water bath to 56 C Preheat Elution Buffer BE to 70 C For centrifugation a centrifuge with a swing out rotor and appropriate buckets capable of reaching 4 000 4 500 x gis required
25. ou process s 5 mL blood sample loading with a single centrifugation step is possible step 3 10 mL blood If processing buffy coat do not use more than 2 mL and add 500 pL PBS to adjust the volume to 10 mL a Proteinase K If cultured cells are used resuspend up to 1 x 10 cells in a final volume of 10 mL PBS Add 10 mL Buffer BQ1 if processing less than 10 mL 10 mL BQ1 blood add one volume of Buffer BQ1 to the samples and vortex the mixture vigorously for 10 s Mix Note Vigorous mixing is important to obtain high yield and purity of DNA 56 C Incubate samples at 56 C for 15 min 15 min Let the lysate cool down to room temperature before proceeding with addition of ethanol The lysate should become brownish during incubation with Buffer BQ1 Increase incubation time with Proteinase K up to 20 min and vortex once or twice during incubation if processing older or clotted blood samples MACHEREY NAGEL 06 2014 Rev 14 21 NucleoSpin Blood XL Adjust DNA binding conditions Add 10 mL ethanol 96 100 if processing less than 10 mL blood add one volume of ethanol to each sample and mix by inverting the tube 10 times Note High local ethanol concentration must be avoided by immediate mixing after addition Be sure that the lysate has cooled down to room temperature about 5 min before loading it onto the columns Loading of hot lysate may lead to diminished yields Bind DNA For each prepar
26. rature by inhibition of omnipresent DNases However EDTA interferes depending on the final concentration with certain downstream applications For optimal performance of isolated DNA in subsequent downstream applications we recommend elution with the supplied elution buffer and storage especially long term at 20 C Several freeze thaw cycles will not interfere with most downstream applications Performance of long range PCR e g gt 10 kbp or detection sensitivity of trace amount of DNA species might be reduced after multiple freeze thaw cycles or prolonged storage of eluted DNA at 4 C or room temperature due to DNA shearing or adsorption to surfaces 350 100 300 80 250 T 7 2 AS A 60 200 zg 57 3 BE 25 150 58 lt 2 40 25 25 ne 100 amp z a N a oO 0 025 05 0 75 1 125 15 1 75 2 Elution volume mL Figure 1 Dependence of DNA yield solid line and concentration dashed line on elution volume Genomic DNA was purified from 10 mL whole blood and eluted using different elution volumes as indicated Highest DNA yield is obtained with 1 5 2 0 mL elution volume Highest DNA concentration is obtained with approximately 0 75 mL elution volume Furthermore yield and concentration may vary as they depend on the kind of sample blood serum plasma type of blood sample human or animal and quality of the samples fresh old frozen clotted etc MACHEREY NAGEL 06 2014 Rev 14 11 Genomic
27. rmation Product REF Pack of NucleoSpin Blood 740951 10 50 250 10 50 250 NucleoSpin Blood L 740954 20 20 NucleoSpin Blood XL 740950 10 50 10 50 NucleoSpin Blood QuickPure 740569 10 50 250 10 50 250 Buffer BQ1 740923 125 mL Buffer B3 740920 100 mL ne 740921 20 mL Bufer BW 740922 100 mL Proteinase K 740506 100 mg RNase aa Fai Collection Tubes 2 mL 740600 1000 Visit www mn net com for more detailed product information 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 MACHEREY NAGEL 06 2014 Rev 14 29 Genomic DNA from blood 6 4 Product use restriction warranty NucleoSpin Blood kit components are intended developed designed and sold FOR RESEARCH PURPOSES ONLY except however any other function of the product being expressly described in original MACHEREY NAGEL product leaflets MACHEREY NAGEL products are intended for GENERAL LABORATORY USE ONLY MACHEREY NAGEL products are suited for QUALIFIED PERSONNEL ONLY MACHEREY NAGEL products shall in any event only be used wearing adequate PROTECTIVE CLOTHING For detailed information please refer to the respective Material Safety Data Sheet of the product MACHEREY NAGEL products shall exclusively be used in an ADEQUATE TEST ENVIRONMENT MACHEREY NAGEL does not assume any responsibility for damages due to improper application of our products in other
28. rotor Vortex mixer Thermal heating block NucleoSpin Blood QuickPure or water bath NucleoSpin Blood L XL Personal protection equipment lab coat gloves goggles 1 3 About this user manual It is strongly recommended reading the detailed protocol sections of this user manual if the NucleoSpin Blood kit is used for the first time Experienced users however may refer to the Protocol at a glance instead The Protocol at a glance is designed to be used only as a supplemental tool for quick referencing while performing the purification procedure All technical literature is available on the internet at www mn net com Please contact Technical Service regarding information about changes of the current user manual compared to previous revisions 8 MACHEREY NAGEL 06 2014 Rev 14 Genomic DNA from blood 2 2 1 Product description The basic principle With the NucleoSpin Blood method genomic DNA is prepared from whole blood cultured cells serum plasma or other body fluids Lysis is achieved by incubation of whole blood in a solution containing large amounts of chaotropic ions in the presence of Proteinase K Appropriate conditions for binding of DNA to the silica membrane of the corresponding NucleoSpin Blood Columns are achieved by addition of ethanol to the Iysate The binding process is reversible and specific to nucleic acids Washing steps efficiently remove contaminations With the NucleoSpin Bloo
29. t need to be labeled with H and P phrases up to 125 mL or 125 g Mindergef hrliche Eigenschaften m ssen bis 125 mL oder 125 g nicht mit H und P S tzen gekennzeichnet werden Component Hazard contents GHS symbol Hazard Precaution phrases phrases Inhalt Gefahrstoff GHS Symbol H S tze P S tze B3 Guanidine hydrochloride Warning 302 319 280 301 312 36 50 305 351 338 330 Guanidinhydrochlorid 36 50 Achtung 337 313 BQ1 Guanidine hydrochloride Warning 302 315 280 301 312 50 66 319 302 352 Guanidinhydrochlorid 50 66 Achtung 305 351 338 330 332 313 337 313 BW Guanidine hydrochloride Warning 226 302 210 233 280 36 50 isopropanol 319 301 312 20 50 305 351 338 330 Guanidinhydrochlorid 36 50 Achtung 337 313 403 235 Isopropanol 20 50 Proteinase K Proteinase K lyophilized Danger 315 319 261 280 302 352 Proteinase K lyophilisiert Gefahr 334 335 304 340 305 351 338 312 D 332 313 337 313 342 311 403 233 Hazard phrases H 226 Flammable liquid and vapour Fl ssigkeit und Dampf entz ndbar H 302 Harmful if swallowed Gesundheitssch dlich bei Verschlucken H 315 Causes skin irritation Verursacht Hautreizungen H 317 May cause an allergic skin reaction Kann allergische Hautreaktionen verursachen H 319 Causes serious eye irritation Verursacht schwere Augenreizung H 334 May cause allergy or asthma symptoms or breathing difficulties if inhaled Kann
30. uboptimal performance of genomic DNA in enzymatic reactions Proteinase K digestion is not optimal Do not add Proteinase K directly to lysis buffer Incubate for at least 15 20 min at 56 C 70 C RNA in sample If RNA free DNA is desired add 20 uL RNase A solution 20 mg mL before addition of lysis buffer Old or clotted blood samples processed For isolation of DNA from older or clotted blood samples we recommend prolonging Proteinase K incubation to 30 min and vortexing several times during this step Especially for NucleoSpin Blood L XL with troublesome blood samples performance can be improved by the following steps First incubate the lysate for 10 15 min at room temperature Incubate for 15 min at the recommended 56 C afterwards Clear lysate before addition of ethanol It is recommended performing a short centrifugation step of about 30 60 s after the lysis of the sample material before addition of ethanol in order to pellet non lysed clumps In case of difficult blood samples it might happen that the washing steps with ethanolic Buffer BQ2 are not sufficient to remove all contamination An additional wash step with a buffer including chaotropic salt is recommended for example water BQ1 ethanol mix 1 1 1 Afterwards the washing step with ethanolic Buffer BQ2 should be performed to completely remove the chaotropic salt of the wash buffer Carry over of ethanol Be sure to remove all of ethanolic Buffer B5
31. utions and storage conditions see section 3 Composition of Elution Buffer BE 5 mM Tris HCl pH 8 5 6 MACHEREY NAGEL 06 2014 Rev 14 Genomic DNA from blood 1 1 Kit contents continued NucleoSpin Blood QuickPure 10 preps 50 preps 250 preps REF 740569 10 740569 50 740569 250 Lysis Buffer BQ1 13 mL 13 mL 60 mL Wash Buffer BQ2 7 mL 7 mL 2x20 mL Concentrate Elution Buffer BE 13 mL 13 mL 60 mL Proteinase K lyophilized 6 mg 30 mg 2x75 mg Proteinase Buffer PB 1 8 mL 1 8 mL 8 mL NucleoSpin Blood QuickPure Columns dark 10 50 250 red rings plus Collection Tubes Collection Tubes 2 mL 10 50 250 User manual 1 1 1 For preparation of working solutions and storage conditions see section 3 Composition of Elution Buffer BE 5 mM Tris HCl pH 8 5 MACHEREY NAGEL 06 2014 Rev 14 7 Genomic DNA from blood 1 2 Reagents consumables and equipment to be supplied by user Reagents 96 100 ethanol Phosphate buffered saline PBS may be required for some samples Consumables 1 5mL microcentrifuge tubes NucleoSpin Blood QuickPure 15 mL NucleoSpin Blood L or 50 mL centrifuge tubes NucleoSpin Blood XL for sample lysis and DNA elution Disposable pipette tips Equipment Manual pipettors Centrifuge for microcentrifuge tubes NucleoSpin Blood QuickPure centrifuge for 15 mL NucleoSpin Blood L or 50 mL NucleoSpin Blood XL centrifuge tubes with a swing bucket
32. warrant the correctness of any of those applications Last updated 07 2010 Rev 03 Please contact MACHEREY NAGEL GmbH amp Co KG Tel 49 24 21 969 270 e mail tech bio mn net com Trademarks Disclaimer NucleoSpin is a trademark of MACHEREY NAGEL GmbH amp Co KG 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 operation MACHEREY NAGEL 06 2014 Rev 14 31
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