NucleoSpin® Dx Blood - MACHEREY
Contents
1. 06 2014 Rev 03 Genomic DNA from blood 6 Appendix 6 1 Troubleshooting Problem Possible cause and suggestions No or poor DNA yield Low concentration of white blood cells in sample The DNA yield depends on the number of white blood cells per sample Blood samples with low white blood cell count yield low DNA amounts Incomplete sample lysis Inhomogeneous blood sample or blood clots within the sample Make sure that blood samples are collected following the instructions of the manufacturer of the blood collection tube Make sure that only blood which can be easily transferred by pipetting is used as sample material If necessary homogenize the blood sample before use Sample not thoroughly mixed with Proteinase K and lysis buffer The mixture has to be vortexed vigorously immediately after addition of Lysis Buffer B3 Proteinase K digestion not optimal Never add Proteinase K directly to Lysis Buffer B3 Reagents not applied properly Prepare buffers and Proteinase K solution according to instructions section 3 Add ethanol to lysate before loading lysate on the column Unappropriate centrifugation Do not extend centrifugation time and speed in step 6 and 8 Only use a short spin to remove droplets from the lid Suboptimal elution of DNA from the column Elution efficiency depends on elution buffer volume For highest elution efficiency use 200 uL elution buffer for highest DNA concen2. 49 0 24 21 969 270 e mail TECH BIO mn net com Last updated 09 2009 Rev 03 Trademarks BD VACUETTE is a trademark of BD NucleoSpin is a trademark of MACHEREY NAGEL GmbH amp Co KG S Monovette is a registered trademark of Sarstedt Vacuette is a trademark of GREINER BIO ONE 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 03 21
3. www mn net com Genomic DNA from blood User manual NucleoSpin Dx Blood ce IVD REF aal Cli MACHEREY NAGEL In Vitro Diagnostic Medical Device 740899 50 740899 250 MACHEREY NAGEL GmbH amp Co KG D 52355 D ren Tel 49 0 2421 969 0 June 2014 Rev 03 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 6 1 3 About this user manual 6 2 Product description 7 2 1 Intended use 7 2 2 Product use limitations 7 2 3 Quality control 7 2 4 Introduction and kit specifications 8 2 5 Elution procedures 9 3 Storage conditions and preparation of working solutions 10 4 Safety instructions 11 5 Genomic DNA purification with NucleoSpin Dx Blood 13 5 1 Protocol at a glance 14 5 2 Procedure 15 6 Appendix 17 6 1 Troubleshooting 17 6 2 Ordering information 19 6 3 Product use restriction warranty 20 MACHEREY NAGEL 06 2014 Rev 03 3 Genomic DNA from blood 1 Components 1 1 Kit contents NucleoSpin Dx Blood 50 preps 250 preps REF 740899 50 740899 250 Buffer B3 sur 83 15 mL 60 mL Wash Buffer BW sur Bw 30 mL 150 mL Wash Buffer B5 eur B5 conc 12 mL 50 mL Concentrate Elution Buffer BE BUF BE 13 mL 60 mL Proteinase Buffer PB BUF PB 1 8 mL 8 mL Proteinase K Proteinase K lyophilized 20 19
4. heating block or water bath to 70 C Equilibrate the blood samples to room temperature 18 25 C Make sure that the samples are mixed well If a precipitate has formed in Lysis Buffer B3 or Buffer BW incubate the buffer at 70 C until the precipitate is dissolved Generally do not mix reagents and columns from different kits and lots Equilibrate Elution Buffer BE to room temperature 18 25 C Do not add Proteinase K solution directly to Lysis Buffer B3 Proteinase K has to be mixed with the blood sample before addition of Buffer B3 All centrifugation steps should be carried out at room temperature 18 25 C MACHEREY NAGEL 06 2014 Rev 03 13 NucleoSpin Dx Blood 5 1 Protocol at a glance Supplemental protocol overview Carefully read the detailed protocol section 5 2 before starting the procedure Lyse blood 1 25 uL Proteinase K samples 2 200 uL blood 3 200 uL B3 mix y 4 RT 5 min 5 70 C 10 min mix S 6 2 000 x g 1 s Adjust DNA 7 210 uL ethanol mix binding conditions 8 2 000 x g 1 s Bind DNA 9 Load lysate 10 11 000 x g 1 min 11 Transfer the NucleoSpin Dx Blood Column to a new Collection Tube Wash silica 12 500 uL BW membrane 13 11 000 x g 1 min 14 Transfer the NucleoSpin Dx Blood Column to a new Collection Tube 15 600 uL B5 16 11 000 x g 1 min Dry silica 17 Transfer the NucleoSpin Dx Blood Column membrane to a new Collection Tube eS 18 11 000 x g 1 mi
5. Mindergefahrliche 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 Guanidinhydrochlorid 36 50 Achtung 330 337 313 BW Guanidine hydrochloride Warning 226 302 210 233 280 36 50 isopropanol 319 301 312 20 50 305 351 338 Guanidinhydrochlorid 36 50 Achtung 330 337 313 Isopropanol 20 50 403 235 Proteinase K Proteinase K Iyophilized Danger 334 261 304 340 Proteinase K lyophilisiert lt gt Gefahr 342 311 Hazard phrases H 226 Flammable liquid and vapour Fl ssigkeit und Dampf entz ndbar H 302 Harmful if swallowed Gesundheitssch dlich bei Verschlucken H 319 Causes serious eye irritations Verursacht schwere Augenreizung H 334 May cause allergy or asthma symptoms or breathing difficulties if inhaled Kann bei Einatmen Allergie asthmaartige Symptome oder Atembeschwerden verursa chen Precaution phrases P 210 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 P 233 Keep container tightly closed Beh lter dicht verschlossen halten P 261 Avoid breathing dust Einatmen von Staub vermeid
6. NucleoSpin Dx Blood kit wear suitable protective clothing e g lab coat disposable gloves and protective goggles For more information consult the appropriate Material Safety Data Sheets MSDS available online at http www mn net com msds Caution Guanidine hydrochloride in Lysis Buffer B3 and Wash Buffer BW can form highly reactive compounds when combined with bleach Thus do not add bleach or acidic solutions directly to the sample preparation waste The waste generated with the NucleoSpin Dx Blood kit has not been tested for residual infectious material A contamination of the liquid waste with residual infectious material is highly unlikely due to strong denaturing lysis buffer and Proteinase K treatment but it cannot be excluded completely Therefore liquid waste must be considered infectious and should be handled and discarded according to local safety regulations 12 MACHEREY NAGEL 06 2014 Rev 03 Genomic DNA from blood 5 Genomic DNA purification with NucleoSpin Dx Blood The procedure below provides instructions for processing a single blood sample However several samples can be processed at the same time the number depends on the capacity of the microcentrifuge used Before starting the preparation Check if Buffer B5 and Proteinase K were prepared according to section 3 Check that 96 100 ethanol denatured or non denatured is available to adjust DNA binding conditions Setan incubator e g
7. dade catalogo ficiente m dico strugdes tempe para n para dia de utili ratura ensaios gn stico za o in vitro MACHEREY NAGEL 06 2014 Rev 03 5 Genomic DNA from blood O y tor REF Y al vo a f x SV teran Anv nd Lot num Katalog Racker Tillver Medicin Se Tempe Irritan vand ej for mer nummer till n kare tekniska handha raturbe derende antal produk vande grans tester ter f r beskriv ning in vitro ningen diagnos tik 1 2 Reagents consumables and equipment to be supplied by user Reagents 96 100 ethanol to adjust DNA binding conditions and to prepare Wash Buffer B5 Consumables Disposable pipet tips aerosol barrier pipet tips are recommended to avoid cross contamination Equipment Manual pipettors Centrifuge for microcentrifuge tubes Vortex mixer Thermal heating block or water bath for samples lysis at 70 C Personal protection equipment e g lab coat gloves goggles 1 3 About this user manual It is strongly recommended that first time users of the NucleoSpin Dx Blood kit read the detailed protocol sections of this user manual 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 perfo
8. diagnostic result It is the sole responsibility of the user to use and validate the kit in conjunction with a downstream in vitro diagnostic assay 2 2 Product use limitations The NucleoSpin Dx Blood kit is not for use with tissue or stool samples cell free body fluids such as plasma serum urine or cerebrospinal fluid The kit performance has not been evaluated with buffy coat cultured or isolated cells swabs dried blood spots and viral DNA The kit is also not specified for the isolation and purification of bacterial fungal or parasite nucleic acids 2 3 Quality control In accordance with MACHEREY NAGEL s Quality Management System each lot of NucleoSpin Dx Blood kit is tested against predetermined specifications to ensure consistent product quality MACHEREY NAGEL 06 2014 Rev 03 7 Genomic DNA from blood 2 4 Introduction and kit specifications NucleoSpin Dx Blood is based on well established NucleoSpin silica membrane technology and provides an easy way to isolate genomic DNA from 200 uL of whole blood samples Purified DNA is ready to use for downstream PCR amplification The NucleoSpin Dx Blood procedure is based on a series of simple steps First the blood samples are lysed in the presence of chaotropic salts and Proteinase K The genomic DNA in the lysate is then bound to a NucleoSpin Dx Blood Column Subsequently the membrane with bound nucleic acids is washed and finally highly pure genom
9. new sample material Place the NucleoSpin Dx Blood Column into a new Collection Tube 2 mL provided and discard the Collection Tube with flow through from the previous step Open the NucleoSpin Dx Blood Column and add 500 uL Buffer BW to the column Close the lid Note Make sure that residual lysate is washed away with Buffer BW Centrifuge 1 min at 11 000 x g MACHEREY NAGEL 06 2014 Rev 03 15 NucleoSpin Dx Blood 14 15 16 17 18 19 20 21 Place the NucleoSpin Dx Blood Column into a new Collection Tube 2 mL provided and discard the Collection Tube with flow through from the previous step Open the NucleoSpin Dx Blood Column and add 600 pL Buffer B5 to the column Close the lid Note Make sure that residual wash buffer from the previous step is washed away with Buffer B5 Centrifuge 1 min at 11 000 x g Place the NucleoSpin Dx Blood Column into a new Collection Tube 2 mL provided and discard the Collection Tube with flow through from the previous step Centrifuge 1 min at 11 000 x g Residual ethanol is removed during this step Place the NucleoSpin Dx Blood Column in a clean Elution Tube 1 5 mL provided and discard the Collection Tube from the previous step Open the NucleoSpin Dx Blood Column and add 50 200 uL Buffer BE directly onto the center of the membrane Centrifuge 1 min at 11 000 x gto elute the DNA from the column 16 MACHEREY NAGEL
10. with 50 to 200 uL Elution Buffer BE Overall DNA yield increases with increasing elution volume whereas the DNA concentration decreases see Figure 1 Typically up to 10 uL of the eluate can be used as template in a 50 uL PCR mix without affecting PCR performance It is recommended storing eluted DNA at 20 C Several freeze thaw cycles will not interfere with most downstream applications 6 0 100 so _ 55 2 D 2 60 2 5 0 40 lt zZ A 45 8 20 q z a 4 0 i l 0 0 50 100 150 200 Elution volume pl Figure 1 Impact of elution volume on overall DNA yield dashed line and concentration solid line Elution was performed with 50 100 and 200 uL MACHEREY NAGEL 06 2014 Rev 03 9 Genomic DNA from blood 3 Storage conditions and preparation of working solutions Attention Check all components for damages after receiving the kit If kit contents like buffer bottles or blister packages are damaged contact MACHEREY NAGEL technical support and customer service or your local distributor Do not use damaged kit components Upon arrival the NucleoSpin Dx Blood kit should be stored at room temperature 18 25 C It is NOT required to open the kit on delivery and remove individual components for separate storage NucleoSpin Dx Blood Columns can be used until the expiration date specified on the kit box Before starting the NucleoSpin Dx Blood protocol prepare the follo
11. en MACHEREY NAGEL 06 2014 Rev 03 11 Genomic DNA from blood P 280 Wear protective gloves eye protection Schutzhandschuhe Augenschutz tragen P 301 312 IF SWALLOWED Call a POISON CENTER doctor if you feel unwell BEI VERSCHLUCKEN Bei Unwohlsein GIFTINFORMATIONSZENTRUM Arzt anrufen P 304 340 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 P 305 351 338 IF IN EYES Rinse cautiously with water for several minutes Remove contact lenses if present and easy to do Continue rinsing Bei Kontakt mit den Augen Einige Minuten lang behutsam mit Wasser sp len Vorhandene Kontaktlinsen nach M glichkeit entfernen Weiter sp len P 330 Rinse mouth Mund aussp len P 337 313 If eye irritation persists Get medical advice attention Bei anhaltender Augenreizung Arztlichen Rat einholen rztliche Hilfe hinzuziehen P 342 311 If experiencing respiratory symptoms Call a POISON CENTER doctor Bei Symptomen der Atemwege GIFTINFORMATIONSZENTRUM Arzt anrufen P 403 235 Store in a well ventilated place Keep cool An einem gut bel fteten Ort lagern Kuhl halten 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 When working with the
12. g the DNA If the filling level of Wash Buffer B5 flow through after the second wash reaches the column outlet for any reason discard flow through place the column back into the Collection Tube and centrifuge again DNA eluates may contain traces of ethanol However no decrease in PCR performance was observed using DNA eluate of up to 20 of the PCR final volume as template e g using 4 uL from 100 uL eluate as template in a 20 uL PCR The maximum percentage of template volume in a PCR may vary depending on the robustness of the PCR system and has to be determined by the user 18 MACHEREY NAGEL 06 2014 Rev 03 Genomic DNA from blood Problem Possible cause and suggestions Suboptimal Contamination of DNA with inhibitory substances performance If preparing DNA from old or clotted blood samples make of genomic sure that only blood is used as sample material which can DNA in be easily transferred by pipetting If necessary homogenize enzymatic the blood sample before use reactions continued 6 2 Ordering information Product REF Pack of NucleoSpin Dx Blood 740899 50 250 50 250 NucleoSpin Dx Virus 740895 50 50 Visit www mn net com for more detailed product information MACHEREY NAGEL 06 2014 Rev 03 19 Genomic DNA from blood 6 3 Product use restriction warranty The NucleoSpin Dx Blood Kit is a generic system for the isolation and purification of genomic DNA from human whole b
13. ic DNA is eluted Samples The kit can be used with 200 uL fresh or frozen human whole blood treated with EDTA citrate or heparin from common blood collection systems Cryoprecipitates formed during thawing of frozen samples may clog the NucleoSpin Dx Blood Column If such precipitates are visible avoid aspirating them when loading the lysate to the binding column Typically 200 uL human whole blood will yield 3 5 ug genomic DNA depending on the white blood cell count of the sample A selection of suitable blood collection devices is shown below Table 1 Selection of suitable blood collection systems Blood collecting system Manufacturer S Monovette Li Heparin Sarstedt S Monovette EDTA Sarstedt S Monovette Citrat Sarstedt VACUETTE EDTA GREINER BIO ONE BD VACUTAINER K2E BD K3 EDTA DELTA LAB K2 EDTA APTACA 8 MACHEREY NAGEL 06 2014 Rev 03 Genomic DNA from blood Table 2 Kit specifications at a glance Parameter NucleoSpin Dx Blood Sample material Fresh and frozen human whole blood treated with EDTA citrate or heparin from common blood collection systems Sample volume 200 uL Typical DNA yield 3 5 Hg depending on white blood cell count Typical DNA quality Ratio Aago Aaso 1 7 1 9 Ratio A s0 Azao 1 8 2 3 Elution volume 50 200 uL Typical DNA concentration 40 60 ng pL Processing Centrifugation 2 5 Elution procedures DNA is eluted from the NucleoSpin Dx Blood Columns
14. lood samples for subsequent in vitro diagnostic purposes The kit is designed to be used with downstream applications employing enzymatic amplification and detection of DNA e g PCR Any and all diagnostic results generated using the DNA isolated with the NucleoSpin Dx Blood kit in conjunction with a diagnostic assay should be interpreted with regard to additional clinical or laboratory findings The NucleoSpin Dx Blood Kit does not provide a diagnostic result It is the sole responsibility of the user to use and validate the kit in conjunction with a downstream in vitro diagnostic assay ONLY MACHEREY NAGEL products specially labeled as IVD are suitable for In vitro diagnostic use NucleoSpin Dx Blood kit is intended for use by professional users such as technicians and physicians experienced in and trained in molecular biological techniques including experience with whole blood samples and DNA isolation For safety instructions please refer to the respective chapter in the user manual NucleoSpin Dx Blood kit shall exclusively be used in an adequate test environment i e a suitable laboratory setting The respective user is liable for any and all damages resulting from application of the NucleoSpin Dx Blood kit for use deviating from the intended use as specified in the user manual This MACHEREY NAGEL product is shipped with documentation stating specifications and other technical information MACHEREY NAGEL warrants to meet the sta
15. n 14 MACHEREY NAGEL 06 2014 Rev 03 NucleoSpin Dx Blood Elute DNA 19 Transfer the NucleoSpin Dx Blood Column to an Elution Tube 20 50 200 uL BE 21 11 000 x g 1 min 5 2 Procedure 10 11 12 13 Pipette 25 uL Proteinase K solution into a Lysis Tube 1 5 mL provided Add 200 uL blood sample into the Lysis Tube Mix Add 200 uL Buffer B3 to the Lysis Tube close the lid and mix by pulse vortexing vigorously for 10 s Do not premix Buffer B3 and Proteinase K Incubate at room temperature 18 25 C for 5 min 1 min Incubate the Lysis Tube at 70 C 2 C for 10 min 1 min After incubation mix by pulse vortexing vigorously for 5 s Briefly centrifuge Lysis Tube approx 1 s at 2 000 x g to remove drops from the lid short spin only Add 210 uL ethanol 96 100 to the sample Close the lid and mix by pulse vortexing for 5 s Make sure that the ethanol and the lysate is mixed well Briefly centrifuge Lysis Tube approx 1 s at 2 000 x g to remove drops from the lid short spin only Carefully load the entire lysate to the NucleoSpin Dx Blood Column placed in a Collection Tube and close the lid Centrifuge 1 min at 11 000 x g If the lysate is not completely drawn through the membrane repeat the centrifugation at higher g force 15 000 20 800 x g for 1 min If the lysate still does not pass the membrane completely discard the sample and repeat the isolation with
16. nte sitivo tare le tempe zare logo ficiente medico istruzio ratura per n diagno ni per saggi stico in Puso vitro FR Nepas Utiliser Code du R f Contenu Fabri Dis Consul Limites Irritante r utiliser jusque lot rence du suffisant cant positif ter les de catalo pour n medical instruc temp gue tests de dia tions rature gnostic d utilisa in vitro tion NL Niet Houd Lot num Cata Inhoud Fabri Medisch Raad Tempe Irritie opnieuw baar tot mer logus vol kant hulpmid pleeg de ratuurli rend gebrui nummer doende del voor ge miet ken voor n in vitro bruiks testen diagnos aanwij tiek zing DA Maikke Holdbar Lotnum Katalog Inde Produ Medi Se Tempe Lokalirri genbru til mer nummer holder cent cinsk brugsan raturbe terende ges tilsttraek udstyr til visning gr ns keligt til in vitro ning n test diagno stik EL Mnv Huego Agududc AguOudc Meo Kata In Vitro Zuu ov Mego AtaBow H VETE un a Tlagti xatad yOpevo oxeva Atayvo hevte te Qropot ttxd ena va AnEns das you emagx s otis OTLA TL OeQuo Ansturm Y LOL v Iatgo o nyles xoaotac xonon eEet texvoho yorjons OES y TQOLOV PT Nao reu Prazo C digo Refer n Conte Fabri Dispo Consulte Limites Irritante tilizar de vali do lote cia de do su cante sitivo as in de
17. ood 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 warrant the correctness of any of those applications Please contact MACHEREY NAGEL Germany Tel
18. rming 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 6 MACHEREY NAGEL 06 2014 Rev 03 Genomic DNA from blood 2 Product description 2 1 Intended use The NucleoSpin Dx Blood kit is a generic system for the isolation and purification of genomic DNA from human whole blood samples for subsequent in vitro diagnostic purposes The kit can be used with fresh and frozen human whole blood treated with EDTA citrate or heparin from common blood collection systems The kit is designed to be used with any downstream application employing enzymatic amplification and detection of DNA e g PCR Any diagnostic results generated using the DNA isolated with the NucleoSpin Dx Blood kit in conjunction with an in vitro diagnostic assay should be interpreted with regard to additional clinical or laboratory findings To minimize irregularities in diagnostic results suitable controls for downstream applications e g extraction controls positive negative controls should be used The NucleoSpin Dx Blood kit is intended for use by professional users such as technicians and physicians experienced and trained in molecular biological techniques including experience with whole blood samples and DNA isolation The NucleoSpin Dx Blood kit does not provide a
19. sad NucleoSpin Dx Blood Dx Blood Columns 50 250 Columns red rings plus Collection Tubes Collection Tubes 2 mL 3x50 3 x 250 Lysis Tubes 1 5 mL 50 5x50 Elution Tubes 1 5 mL Elution Tubes 50 5x50 User manual BH 1 1 For preparation of working solutions and storage conditions see section 3 Composition of Elution Buffer BE 5 mM Tris HCI pH 8 5 4 MACHEREY NAGEL 06 2014 Rev 03 Genomic DNA from blood O y Lor REF Y al vo a Y x EN Do not Use by Batch Cata Contains Manuf In Vitro Consult Tempe Irritant reuse code logue sufficient acturer Diag Instruc rature number for lt n gt nostic tions for limitation tests Medical use Device DE Nicht zur Ver Char Bestell Inhalt Herstel In Ge Tempe Reizend Wieder wendbar genbe nummer ausrei ler vitro Di brauchs ratur verwen bis zeich chend agnosti anwei begren dung nung f r lt n gt kum sung zung Tests beach ten ES No reuti Fecha C digo N mero Con Fabri Pro Consul L mite Irritante lizar de cadu de lote de cat tenido cante ducto te las de cidad logo suficien sanitario instruc tempe te para para ciones ratura lt n gt diagn s de uso ensayos tico in vitro IT Non Utilizza Codice Numero Conte Fabbri Dispo Consul Limiti di Irritant riutiliz re entro del lotto dicata nuto suf ca
20. ted 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 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 consequential or special including but not limited to loss of use revenue or profit whether based upon 20 MACHEREY NAGEL 06 2014 Rev 03 Genomic DNA from bl
21. tration use 50 uL elution buffer MACHEREY NAGEL 06 2014 Rev 03 17 Genomic DNA from blood Problem Possible cause and suggestions Clogged DNA binding column Inhomogeneous blood sample Cryoprecipitate formed during thawing of frozen samples may clog the NucleoSpin Dx Blood Column If such precipitates are visible avoid aspirating them when loading the lysate to the binding column Precipitates may also form in fresh blood samples Make sure that the samples are mixed well If the column clogs during the DNA binding step repeat the centrifugation at higher g force 15 000 20 800 x g for 1 min Poor DNA quality Reagents not applied properly e Prepare buffers and Proteinase K solution according to instructions section 3 Add ethanol to lysate and mix before loading them on columns Incomplete sample lysis Sample not thoroughly mixed with Proteinase K solution and lysis buffer The mixture has to be vortexed vigorously immediately after addition of lysis buffer Proteinase K digestion not optimal Do not add Proteinase K directly to Lysis Buffer B3 Old or clotted blood samples processed Make sure that only blood is used as sample material which can be easily transferred by pipetting If necessary homogenize the blood sample before use Suboptimal performance of genomic DNA in enzymatic reactions Carry over of ethanol Be sure to remove all of ethanolic Buffer B5 before elutin
22. wing Wash Buffer B5 Add the indicated volume of ethanol 96 100 see table below or on the bottle 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 until the expiration date Lyophilized Proteinase K can be stored at room temperature 18 25 C until the expiration date without decrease in performance Before first use of the kit add the indicated volume of Proteinase Buffer PB to dissolve lyophilized Proteinase K Reconstituted Proteinase K should be stored at 20 C for up to 6 months but only until the expiration date During storage especially at low temperatures a white precipitate may form in Buffer B3 and Buffer BW Such precipitates can be easily dissolved by incubating the bottle at 70 C for 5 min before use NucleoSpin Dx Blood 50 preps 250 preps REF 740899 50 740899 250 Wash Buffer B5 12 mL 50 mL Concentrate Add 48 mL ethanol Add 200 mL ethanol Proteinase K 30 mg 2x 75 mg Add 1 35 mL Add 3 35 mL Proteinase Buffer Proteinase Buffer to each vial 10 MACHEREY NAGEL 06 2014 Rev 03 Genomic DNA from blood 4 Safety instructions The following components of the NucleoBond kits contain hazardous contents Wear gloves and goggles and follow the safety instructions given in this section GHS classification Only harmful features need not be labeled with H and P phrases up to 125 mL or 125 g
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