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1. 1 Prepare sample Resuspend up to 10 cells in a final volume of 80 pL Buffer T1 2 Pre lyse sample Add 8 uL Proteinase K solution and mix by vortexing 2x5s Incubate at 56 C for 10 min Adjust the thermal heating block temperature to 70 C at the end of the incubation for the following step If processing several samples Proteinase K and Buffer T1 may be premixed directly before use Do never mix Buffer T1 and Proteinase K more than 10 15 min before addition to the sample Proteinase K tends to self digestion in Buffer T1 without substrate 3 Lyse sample Add 80 uL Buffer B3 vortex 2x5s and incubate at 70 C for 5 min Vortex briefly at the end of the incubation Optional Adjust the thermal heating block temperature to 90 C for the last step of the protocol Let the lysate cool down to room temperature A white precipitate may form in the lysate upon addition of Buffer B3 especially if very small samples are used Precipitates will dissolve during the incubating step at 70 C If insoluble particles are visible after the heat incubation centrifuge for 5 min at high speed e g 11 000 xg and transfer the supernatant to a new microcentrifuge tube not provided y 80 uL T1 8 uL Proteinase K 56 C 10 min 80 uL B3 70 C 5 min 18 MACHEREY NAGEL 02 2012 Rev 04 NucleoSpin Tissue XS Adjust binding conditions Add 80 uL ethanol 96 100 to the lysate and mix by vo
2. a Incubation time min gt 65 70 75 80 85 90 95 Incubation temperature C Figure 2 Removal of residual ethanol from the elution fraction by heat treatment In order to obtain highest PCR sensitivity a heat incubation of the eluate is recommended Heat incubation may be performed at temperatures of 70 90 C ina heat block with or without shaking Effective conditions temperature time and shaking rate for ethanol removal can be read from the diagram an initial volume of 20 uL will evaporate to 12 14 uL during the incubation shown 10 MACHEREY NAGEL 02 2012 Rev 04 Genomic DNA from tissue 3 Storage conditions and preparation of working solutions Attention Buffer B3 contains chaotropic salt and detergents Wear gloves and goggles CAUTION Buffer B3 contains 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 Upon storage especially at low temperatures a white precipitate may form in Buffers T1 or Buffer B3 Such precipitates can be easily dissolved by incubating the bottle at 50 70 C before use Before starting any NucleoSpin Tissue XS protocol prepare the following e Wash Buffer B5 Add the indicated volume see bottle or table below of ethanol
3. Genomic DNA from tissue User manual NucleoSpin Tissue XS February 2012 Rev 04 MACHEREY NAGEL MN Genomic DNA from tissue Protocol at a glance Rev 04 1 Prepare sample NucleoSpin Tissue XS Up to 2 5 mg tissue 2 Pre lyse sample 80 uL T1 8 uL Proteinase K 56 C 1 4 h 3 Lyse sample 80 uL B3 70 C 5 min 4 Adjust binding conditions 80 uL ethanol 5 Bind DNA Load lysate 11 000 x g 1 min 6 Wash silica 50 uL B5 membrane 1stwash 11 000 x g 1 min 50 uL B5 cS 2 wash 11 000 x g 2 min 7 Elute DNA 20 uL BE 11 000 x g e gt 1 min 8 Optional Optional Remove residual ethanol 90 C 8 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 tissue Table of contents 1 Components 1 1 Kit contents 1 2 Reagents consumables and equipment to be supplied by user 1 3 About this user manual 2 Product description 2 1 The basic principle 2 2 Kit specifications 2 3 Handling of sample material 2 4 Elution procedures 2 5 Removal of residual traces of ethanol for highest sensitivity in downstream applications 3 Storage conditions and preparation of working solutions 4 Safety instructions 4 1 Risk and safety phrases 4 2 GHS classification 5 Protocols 5 1 Protocol for human or animal tissue 5
4. Add 80 uL Buffer T1 and 8 uL Proteinase K solution Mix by vortexing 2 5 s and incubate for approximately 1 4 h or overnight at 56 C The optimal incubation time may vary depending on sample type and amount 3 Lyse sample Add 80 uL Buffer B3 vortex 2x5 s and incubate at 70 C for 5 min Vortex briefly after the incubation Let the lysate cool down to room temperature 4 Adjust binding conditions Add 80 uL ethanol 96 100 to each sample and mix by vortexing 2 x 5s Spin down briefly to clear the lid Proceed with step 5 Bind DNA of the standard protocol see section 5 1 28 MACHEREY NAGEL 02 2012 Rev 04 NucleoSpin Tissue XS 5 7 Protocol for blood samples Before starting the preparation Check if Wash Buffer B5 and Proteinase K were prepared according to section 3 Adjust thermal heating block temperature to 56 C and equilibrate sample to room temperature 18 25 C 1 Prepare sample Not necessary 2 Lyse blood samples Pipette 8 uL Proteinase K solution and up to 20 uL blood into a 1 5 mL microcentrifuge tube not provided Add 60 uL Buffer T1 Note For larger blood samples 20 30 uL add 16 uL Proteinase K and add 120 uL Buffer T1 The volumes of Buffer B3 and ethanol have to be increased to 160 uL during the following procedure 3 Lyse sample Add 80 uL Buffer B3 to the sample and vortex the mixture vigorously 10 20 s Incubate samples at 70 C for 10 15 min
5. 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 Tissue XS 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 tech bio mn net com regarding information about changes of the current user manual compared to previous revisions MACHEREY NAGEL 02 2012 Rev 04 5 Genomic DNA from tissue 2 Product description 2 1 The basic principle The NucleoSpin Tissue XS kit is designed for the efficient isolation of genomic DNA from small samples of different kinds of cells and tissues such as laser microdissected samples small amounts of blood or dried blood spots and forensic samples Due to a special funnel design the NucleoSpin Tissue XS Columns allow very small elution volumes 5 30 uL which results in highly concentrated DNA Lysis is achieved by incubation of the sample material in a Proteinase K supplemented lysis buffer Appropriate conditions for binding of the DNA to a silica membrane are created by adding ethanol The mixture is applied to the NucleoSpin Tissue XS Column and DNA bind
6. to the low sensitivity of the absorption between A260 measurement When performing absorption measure quantification ments close to the detection limit of the photometer the values and PCR measurement may be influenced by minor amounts of quantification silica abrasion In order to prevent incorrect As quan values tification of small DNA amounts centrifuge the eluate for 30 s at gt 11 000 x g and take an aliquot for measurement without disturbing any sediment Alternatively use a silica abrasion insensitive DNA quantification method e g PicoGreen fluorescent dye Measurement not in the range of photometer detection limit In order to obtain a significant Azgo Azgo ratio it is necessary Unexpected that the initially measured Ags and As values are Azeo Azgo ratio significantly above the detection limit of the photometer used An Aggy value close to the background noise of the photometer will cause unexpected Asgo Aaso ratios 30 MACHEREY NAGEL 02 2012 Rev 04 Genomic DNA from tissue 6 2 Ordering information Product REF Pack of NucleoSpin Tissue XS 740901 10 50 250 10 50 250 NucleoSpin Tissue 740952 10 50 250 10 50 250 NucleoSpin FFPE DNA 740980 10 50 250 10 50 250 Buffer T1 740940 25 25 mL Buffer B3 740920 100 mL ce 740921 20 mL Buffer BE 740306 100 100 mL Proteinase K 740506 100 mg RNase A 740505 50 50 mg 7410505 100 mg NucleoCard 740403 10 100 10 100 NucleoSpin Filters 740606 50 Col
7. uL of n octane or xylene can be used MACHEREY NAGEL 02 2012 Rev 04 21 NucleoSpin Tissue XS Pre lyse sample Add 80 uL Buffer T1 and 8 uL Proteinase K solution and mix by vortexing 2 x 5s Be sure that the sample is completely covered with lysis solution If processing several samples Proteinase K and Buffer T1 may be premixed directly before use Do never mix Buffer T1 and Proteinase K more than 10 15 min before addition to the sample Proteinase K tends to self digestion in Buffer T1 without substrate Incubate at 56 C until complete Iysis is obtained approximately 1 4 h or overnight Vortex occasionally during incubation or use a shaking incubator At the end of the incubation adjust the thermal heating block temperature to 70 C for the following step Lyse sample Add 80 uL Buffer B3 vortex 2x5s and incubate at 70 C for 5 min Vortex briefly at the end of the incubation Optional Adjust the thermal heating block temperature to 90 C for the last step of the protocol Let the lysate cool down to room temperature A white precipitate may form in the lysate upon addition of Buffer B3 especially if very small samples are used Precipitates will dissolve during the incubating step at 70 C If insoluble particles are visible after the heat incubation centrifuge for 5min at high speed e g 11 000xg and transfer the supernatant to a new microcentrifuge tube not provided Adjust bind
8. volume for your individual application Y 054 gt 720 5 21 04 4 Pal 2 Li v p15 Si 034 ae a 2 r g T 2 10 2 ae 5i 0 24 ws 21 ze gt 6 O14 7 0 5 oO 20 uL 10 pL 5 ul Decreasing elution volume Figure 1 Correlation between elution volume and DNA concentration NucleoSpin Tissue XS Columns 8 MACHEREY NAGEL 02 2012 Rev 04 Genomic DNA from tissue 2 5 Removal of residual traces of ethanol for highest sensitivity in downstream applications A reduction of the 20 uL default elution volume will increase the concentration of residual ethanol in the eluate For 20 uL elution volumes a heat incubation of the elution fraction incubate eluate with open lid for 8 min at 90 C is recommended if the eluate comprises more than 20 of the final PCR volume in order to avoid an inhibition of sensitive downstream reactions In this context please mind the remarks below a An incubation of the elution fraction at higher temperatures will increase signal output in PCR This is especially of importance if the template represents more than 20 of the total PCR reaction volume e g more than 4 uL eluate used as template in a PCR reaction with a total volume of 20 uL The template may represent up to 40 of the total PCR reaction volume if the eluate is incubated at elevated temperature as described above A volume of 20 uL used for elution will evaporate to 12 14 uL during a heat incub
9. 1 P 403 233 IF INHALED If breathing is difficult remove to fresh air and keep at rest ina position comfortable for breathing BEI EINATMEN Bei Atembeschwerden an die frische Luft bringen und in einer Position ruhigstellen die das Atmen erleichtert 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 Call a POISON CENTER or doctor physician if you feel unwell Bei Unwohlsein GIFTINFORMATIONSZENTRUM oder Arzt anrufen Rinse mouth Mund aussp len IF skin irritation occurs Get medical advice attention Bei Hautreizung rztlichen Rat einholen rztliche Hilfe hinzuziehen If eye irritation persists Get medical advice attention Bei anhaltender Augenreizung Arztlichen Rat einholen rztliche Hilfe hinzuziehen If experiencing respiratory symptoms Call a POISON CENTER or doctor physician Bei Symptomen der Atemwege GIFTINFORMATIONSZENTRUM oder Arzt anrufen Store in a well ventilated place Keep container tightly closed Beh lter dicht verschlossen 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 14 MACHEREY NAGEL 02 2012 Re
10. 2 Protocol for cultured cells 5 3 Protocol for paraffin embedded tissue 5 4 Protocol for dried blood spots Guthrie cards 5 5 Protocol for buccal swabs 5 6 Protocol for laser microdissected tissue 5 7 Protocol for blood samples 6 Appendix 6 1 Troubleshooting 6 2 Ordering information 6 3 Product use restriction warranty ana A A oN DD 0 11 12 12 13 15 15 18 21 24 26 28 29 30 30 31 31 MACHEREY NAGEL 02 2012 Rev 04 Genomic DNA from tissue 1 Components 1 1 Kit contents NucleoSpin Tissue XS 10 preps 50 preps 250 preps REF 740901 10 740901 50 740901 250 Lysis Buffer T1 5 mL 10 mL 50 mL Lysis Buffer B3 5 mL 10 mL 50 mL Wash Buffer B9 imt 2 mL 6 mL Concentrate Elution Buffer BE 5mL 5mL 15 mL Proteinase K lyophilized 6 mg 20 mg 2x 50mg Proteinase Buffer PB 0 8 mL 1 8 mL 8 mL en NucleoSpin Tissue XS 40 50 250 Columns green rings 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 HCI pH 8 5 4 MACHEREY NAGEL 02 2012 Rev 04 Genomic DNA from tissue 1 2 Reagents consumables and equipment to be supplied by user Reagents 96 100 ethanol Consumables 1 5 mL microcentrifuge tubes for sample lysis and DNA elution Disposable tips Equipment Manual pipettors Centrifuge for microcentrifuge tubes Vortex mixer Thermal heating block
11. 4 Adjust binding conditions Add 80 uL ethanol 96 100 to the lysate and mix by vortexing 2 x 5 s Spin down briefly to clear the lid 5 Bind DNA For each sample place one NucleoSpin Tissue XS Column into a Collection Tube 2 mL Apply the sample to the column Centrifuge for 1 min at 11 000 x g Discard the flow through and place the column into a new Collection Tube 2 mL Proceed with step 6 Wash silica membrane of the standard protocol see section 5 1 MACHEREY NAGEL 02 2012 Rev 04 29 Genomic DNA from tissue 6 Appendix 6 1 Troubleshooting Problem Possible cause and suggestions Low DNA content of the sample The content of DNA depends very much on sample type amount and quality Low DNA yield Sample contains residual cell debris or cells The lysate may have contained residual particular matter Make sure to proceed after the lysis step only with clear lysate before adding ethanol to create binding conditions Column clogging Residual ethanol in eluate Please see the detailed description of removal of residual traces of ethanol in section 2 6 No increase of PCR signal despite of an increased volume of eluate used as template in PCR Silica abrasion from the membrane Due to the typically low DNA content in very small samples and the resulting low total amount of isolated DNA a DNA quantification via Ag absorption measurement is often Discrepancy hampered due
12. 96 100 to 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 Before first use of the kit add the indicated volume see bottle or table below of Proteinase Buffer PB to dissolve lyophilized Proteinase K Proteinase K solution is stable at 20 C for 6 months NucleoSpin Tissue XS 10 preps 50 preps 250 preps REF 740901 10 740901 50 740901 250 Wash Buffer B5 1mL 2mL 6 mL Concentrate Add 4 mL ethanol Add 8 mL ethanol Add 24 mL ethanol Proteinase K 6 mg 20 mg 2x 50 mg Add 260 uL Add 1 mL Add 2 5 mL Proteinase Buffer Proteinase Buffer Proteinase Buffer to each vial MACHEREY NAGEL 02 2012 Rev 04 11 Genomic DNA from tissue 4 Safety instructions The following components of the NucleoSpin Tissue XS kits contain hazardous contents Wear gloves and goggles and follow the safety instructions given in this section 4 1 Risk and safety phrases Component Hazard contents Hazard Risk Safety symbol phrases phrases Inhalt Gefahrstoff Gefahrstoff R S tze S S tze symbol B3 Guanidine hydrochloride x Xn R 22 36 26 39 Guanidinhydrochlorid Proteinase K Proteinase K lyophilized x Xn R 36 37 38 S 22 24 Proteinase K lyophilisiert 42 26 36 37 Risk phrases R 22 Harmful if swallowed Gesundheitssch dlich beim Verschlucken R 36 Irritating to eyes Reizt die Augen R 36 37 38 Irritating to ey
13. GEL 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 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 32 MACHEREY NAGEL 02 2012 Rev 04 Genomic DNA from tissue 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 appeari
14. GN 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 documentation 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 NA
15. Optional Remove residual ethanol Incubate elution fraction with open lid for 8 min at 90 C See section 2 5 for further comments and alternative incubation times and temperatures for a removal of residual ethanol Load lysate 11 000 x g 1 min 50 pL B5 11 000 x g 1 min 50 pL B5 11 000 x g 2 min 20 uL BE 11 000 x g 1 min Optional 8 min 90 C MACHEREY NAGEL 02 2012 Rev 04 23 NucleoSpin Tissue XS 5 4 Protocol for dried blood spots Guthrie cards Before starting the preparation Check if Wash BufferB5 and Proteinase K were prepared according to section 3 Adjust thermal heating block temperature to 56 C and equilibrate sample to room temperature 18 25 C Prepare sample Cut out one dried blood spot Use only blood soaked paper Cut spots into small pieces and place them in a 1 5 mL microcentrifuge tube not provided The area of the dried blood spot should be less than 30 mm corresponds to approximately 20 uL blood Pre lyse sample Add 160 uL Buffer T1 and mix by vortexing for 2 x 5 s Incubate the sample for 10 min at 94 C Subsequently adjust the thermal heating block temperature to 56 C for the following step Let the sample cool down to room temperature Add 16 pL Proteinase K solution Mix by vortexing and spin the sample down briefly Incubate at 56 C for 1 h Vortex occasionally during incubation or use a shaking incubator Adjust the th
16. Spin Tissue XS 5 5 Protocol for buccal swabs Before starting the preparation Check if Wash Buffer B5 and Proteinase K were prepared according to section 3 Adjust thermal heating block temperature to 56 C and equilibrate sample to room temperature 18 25 C Prepare sample Collect the samples with cotton dacron Daigger or C E P swabs Gibco BRL Scrape firmly against the inside for each cheek several times and let the swabs air dry The respective individual should not have consumed food or drink within 30 min before collection of the samples Pre lyse sample Place the dry swab material in 1 5 mL microcentrifuge tubes not provided Add a mixture of 200 400 uL Buffer T1 and 20 40 uL Proteinase K solution Additional amount of Buffer T1 and Proteinase K is required for this application see ordering information section 6 2 The suitable amount of Buffer T1 depends on the actual size of the buccal swab type Be sure that the buccal swab is completely covered with lysis buffer during incubation Mix by vortexing 2 5 s and incubate 10 min at 56 C 2a Separate lysis solution from buccal swabs Alternative A Place a NucleoSpin Filter not provided see ordering information into a Collection Tube 2 mL Transfer the swab tip cut off swab shaft and the remaining solution onto the NucleoSpin Filter Centrifuge for 1 min at 11 000 x g Discard the NucleoSpin Filter Continue with flow th
17. ademark of Molecular Probes Inc 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 02 2012 Rev 04 33
18. ation for 8 min at 90 C If a higher final volume is required please increase the volume of elution buffer for example from 20 uL to 30 uL An incubation of the elution fraction for 8 min at 90 C will denature DNA If non denatured DNA is required for downstream applications other than PCR for example ligation cloning we recommend an incubation for a longer time at a temperature below 80 C as most of the DNA has a melting point above 80 C Suggestion Incubate for 17 min at 75 C The incubation of the eluate at higher temperatures may be adjusted according to Figure 2 The incubation times and conditions shown will reduce an elution volume of 20 uL to about 12 14 uL and will effectively remove traces of ethanol as described above If the initial volume of elution buffer applied to the column is less than 20 uL heat incubation times should be reduced in order to avoid complete dryness If the elution volume is for example 5 uL a heat incubation of the eluate for 2 min at 80 C will adequately remove residual ethanol The maximum percentage of template volume in a PCR reaction may vary depending on the robustness of the PCR system 40 template volume were tested using LightCycler PCR Roche with the DyNAmo Capillary SYBR Green qPCR Kit Finnzymes MACHEREY NAGEL 02 2012 Rev 04 9 Genomic DNA from tissue 25 without shaking t 700 rpm 1400 rpm De oO
19. e Parameter NucleoSpin Tissue XS Technology Silica membrane technology Format Mini spin columns XS design Typical sample size Tissue samples 0 025 10 mg tissue e g laser microdissected fresh frozen Blood samples 1 30 uL fresh or frozen blood Cultured cells 10 10 000 cultured cells Paraffin embedded tissue 0 001 10 mg tissue Guthrie card spots of 15 30 mm 4 4 6 2 mm Buccal swab one Fragment size 200 bp approx 50 kbp Typical yield Typical yields for selected samples are listed below 100 HeLa cells 0 1 0 5 ng DNA 1000 HeLa cells 1 5 ng DNA 10000 HeLa cells 10 50 ng DNA 0 025 mg mouse liver 20 100 ng DNA 0 25 mg mouse liver 200 1000 ng DNA 2 5 mg mouse liver 600 3000 ng DNA A260 A280 1 7 1 9 Elution volume 5 30 uL Preparation time 20 min prep excl lysis Binding capacity 50 ug 2 3 Handling of sample material The NucleoSpin Tissue XS procedure is designed for very small samples and the typical downstream applications are thus very sensitive It is highly recommended performing sampling and DNA purification with special care in order to avoid a contamination of the sample and the purified DNA with unwanted DNA containing material e g fingerprints hair particles aerosol dust Moreover a cross contamination between samples has to be excluded The following precautions are recommended Wear personal protection equipment lab coat gloves gogg
20. ermal heating block to 70 C for the following step Be sure that the sample is completely covered with lyses buffer during incubation If processing several samples Proteinase K and Buffer T1 may be premixed directly before use Do never mix Buffer T1 and Proteinase K more than 10 15 min before addition to the sample Proteinase K tends to self digestion in Buffer T1 without substrate 24 MACHEREY NAGEL 02 2012 Rev 04 NucleoSpin Tissue XS 2a Separate lysis solution from paper pieces Alternative A Place a NucleoSpin Filter not provided see ordering information into a Collection Tube 2 mL Transfer the complete lysate including paper pieces with a1 mL pipette tip onto the NucleoSpin Filter Centrifuge for 1 min at 11 000 x g Discard the NucleoSpin Filter Continue with flow through Alternative B Transfer as much as possible of the lysate solution to a 1 5 mL microcentrifuge tube not provided Discard paper pieces and continue with recovered solution 3 Lyse sample Add 160 uL Buffer B3 vortex 2x 5s and incubate at 70 C for 5 min Vortex briefly after the incubation Let the lysate cool down to room temperature 4 Adjust binding conditions Add 160 uL ethanol 96 100 to the sample and mix by vortexing 2 x 5 s Spin down briefly to clear the lid Proceed with step 5 Bind DNA of the standard protocol see section 5 1 MACHEREY NAGEL 02 2012 Rev 04 25 Nucleo
21. es respiratory system and skin Reizt die Augen Atmungsorgane und die Haut R 42 May cause sensitization by inhalation Sensibilisierung durch Einatmen m glich Safety phrases S22 Do not breathe dust Staub nicht einatmen S 24 Avoid contact with skin Ber hrung mit der Haut vermeiden S 26 In case of contact with eyes rinse immediately with plenty of water and seek medical advice Bei Ber hrung mit den Augen gr ndlich mit Wasser absp len und Arzt konsultieren S 36 37 Wear suitable protective clothing and gloves Bei der Arbeit geeignete Schutzhandschuhe und Schutzkleidung tragen S39 Wear eye face protection Schutzhandschuhe Gesichtsschutz tragen Hazard labeling not neccessary if quantity per bottle below 125 g 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 12 MACHEREY NAGEL 02 2012 Rev 04 Genomic DNA from tissue 4 2 GHS classification Only harmful features need not be labeled with H and P phrases until 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 hydrochlo Warning 302 319 280 301 312 ride 36 50 305 351 338 330 Guanidinhydroc
22. g information and incubate for additional 5 min at room temperature MACHEREY NAGEL 02 2012 Rev 04 15 NucleoSpin Tissue XS Lyse sample Add 80 uL Buffer B3 vortex 2x 5s and incubate at 70 C for 5 min Vortex briefly at the end of the incubation Optional Adjust the thermal heating block temperature to 90 C for the last step of the protocol Let the lysate cool down to room temperature A white precipitate may form in the lysate upon addition of Buffer B3 especially if very small samples are used Precipitates will dissolve during the incubating step at 70 C If insoluble particles are visible after the heat incubation steps centrifuge for 5 min at high speed e g 11 000 x g and transfer the supernatant to a new microcentrifuge tube not provided Adjust DNA binding conditions Add 80 uL ethanol 96 100 to the lysate and mix by vortexing 2 x 5s Spin down briefly to clear the lid Bind DNA For each sample place one NucleoSpin Tissue XS Column into a Collection Tube 2mL Apply the sample to the column Centrifuge for 1 min at 11 000 x g Discard the flow through and place the column into a new Collection Tube 2 mL If the sample is not drawn completely through the matrix repeat the centrifugation step at 11 000 xg Wash silica membrane Add 50 pL Buffer B5 to NucleoSpin Tissue XS Column Centrifuge for 1 min at 11 000 x g It is not necessary to discard the flow t
23. hlorid Achtung 337 313 36 50 Proteinase K Proteinase K Iyophi Danger 315 319 261 280 302 352 lized 0 334 335 304 340 Proteinase K Iyophilisiert Gefahr 305 351 338 312 332 313 337 313 342 311 403 233 Hazard phrases H 302 Harmful if swallowed Gesundheitssch dich bei Verschlucken H 315 Causes skin irritation Verursacht Hautreizungen H 319 Causes serious eye irritation Verursacht schwere Augenreizung H 334 May cause allergy or asthma symptoms or breathing difficulties if inhaled Kann bei Einatmen Allergie asthmaartige Symptome oder Atembeschwerden verursachen H 335 May cause respiratory irritation Kann die Atemwege reizen Precaution phrases P 261 Avoid breathing dust Einatmen von Staub vermeiden P 280 Wear protective gloves eye protection Schutzhandschuhe Augenschutz tragen P 301 312 IF SWALLOWED Call a POISON CENTER or doctor physician if you feel unwell BEI VERSCHLUCKEN Bei Unwohlsein GIFTINFORMATIONSZENTRUM oder Arzt anrufen P 302 352 IF ON SKIN Wash with soap and water P 304 340 IF INHALED Remove victim to fresh air and keep at rest in a position comfort able for breathing BEI EINATMEN An die frische Luft bringen und in einer Position ruhigstellen die das Atmen erleichtert MACHEREY NAGEL 02 2012 Rev 04 13 Genomic DNA from tissue Precaution phrases P 3044341 P 305 351 338 P 312 P 330 P 3324313 P 3374313 P 342431
24. hrough Reuse the Collection Tube 80 uL B3 en 5 min 80 pL ethanol Load lysate 11 000 x g oS s 1 min 50 pL B5 11 000 x g e gt s 1 min 16 MACHEREY NAGEL 02 2012 Rev 04 NucleoSpin Tissue XS Add 50 uL Buffer B5 to the NucleoSpin Tissue XS Column Centrifuge for 2 min at 11 000 x g Discard Collection Tube with flow through Elute DNA Place the NucleoSpin Tissue XS Column in a new 1 5 mL microcentrifuge tube not provided and apply 20 uL Buffer BE directly onto the center of the silica membrane of the column Centrifuge for 1 min at 11 000 x g Elution volume may be varied from approximately 5 30 uL For a correlation of elution volume DNA concentration and DNA amount eluted from the column see section 2 4 2 5 Optional Remove residual ethanol Incubate elution fraction with open lid for 8 min at 90 C See section 2 5 for further comments and alternative incubation times and temperatures for a removal of residual ethanol 50 pL B5 11 000 x g 2 min 20 uL BE 11 000 x g 1 min Optional 8 min 90 C MACHEREY NAGEL 02 2012 Rev 04 17 NucleoSpin Tissue XS 5 2 Protocol for cultured cells Before starting the preparation Check if Wash Buffer B5 and Proteinase K were prepared according to section 3 Adjust thermal heating block temperature to 56 C and equilibrate sample to room temperature 18 25 C
25. ing conditions Add 80 uL ethanol 96 100 to the lysate and mix by vortexing 2 x 5 s Spin down briefly to clear the lid 80 uL Ti 8 uL Proteinase K 56 C 1 4 h or 56 C overnight 80 pL B3 70 C 5 min 80 pL ethanol 22 MACHEREY NAGEL 02 2012 Rev 04 NucleoSpin Tissue XS Bind DNA For each sample place one NucleoSpin Tissue XS Column into a Collection Tube 2mL Apply the sample to the column Centrifuge for 1 min at 11 000 x g Discard the flow through and place the column into a new Collection Tube 2 mL If the sample is not drawn completely through the matrix repeat the centrifugation step at 11 000 xg Wash silica membrane Add 50 uL Buffer B5 to NucleoSpin Tissue XS Column Centrifuge for 1 min at 11 000 x g It is not necessary to discard the flow through Reuse the Collection Tube Add 50 uL Buffer B5 directly onto the membrane of the NucleoSpin Tissue XS Column Centrifuge for 2 min at 11 000 x g Discard Collection Tube with flow through Elute DNA Place the NucleoSpin Tissue XS Column in a new 1 5 mL microcentrifuge tube not provided and apply 20 uL Buffer BE directly onto the center of the silica membrane of the column Centrifuge for 1 min at 11 000 x g Elution volume may be varied from approximately 5 30 uL For a correlation of elution volume DNA concentration and DNA amount eluted from the column see section 2 4 2 5
26. lection Tubes 2 mL 740600 1000 Visit www mn net com for more detailed product information 6 3 Product use restriction warranty NucleoSpin Tissue XS 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 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 MACHEREY NAGEL 02 2012 Rev 04 31 Genomic DNA from tissue DNA RNA PROTEIN purification products of MACHEREY NAGEL are suitable for IN 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 VITRO diagnostic products are expressly marked as IVD on the packaging IF THERE IS NO IVD SI
27. les Use aerosol resistant pipette tips Always change pipette tips between liquid transfers Briefly centrifuge after mixing steps in order to remove droplets from tube lid MACHEREY NAGEL 02 2012 Rev 04 Genomic DNA from tissue 2 4 Elution procedures A high DNA concentration in the elution fraction is of highest importance and desirable for all typical downstream applications This is of particular interest if the total volume of areaction mixture is limited as this in turn limits the possible amount of added DNA Due to a high default elution volume classical DNA clean up kits often result in weakly concentrated DNA if only small samples are processed Such DNA often even requires a subsequent concentration before it can be used for typical downstream applications In contrast to classical kits NucleoSpin Tissue XS allows an efficient elution in a very small volume which results in highly concentrated DNA An elution volume of 20 uL is recommended by default although volumes as small as 5 uL are feasible A reduction of the elution volume from 20 uL to 5 15 uL will increase DNA concentration whereas the total DNA yield is only slightly affected An increase of the elution volume to 30 uL or more will slightly increase total DNA yield but will reduce DNA concentration Figure 1 gives a graphic description of the correlation between elution volume and DNA concentration and will thus help you to find the optimized elution
28. ng 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 Last updated 07 2010 Rev 03 Please contact MACHEREY NAGEL GmbH amp Co KG Tel 49 24 21 969 270 tech bio mn net com Trademarks Dacron is a registered trademark of Daigger DyNAmo is a trademark of Finnzymes Oy LightCycler is a trademark of a member of the Roche Group NucleoSpin is a registered trademark of MACHEREY NAGEL GmbH amp Co KG PicoGreen is a registered trademark of Molecular Probes Inc SYBR is a registered tr
29. ptional Incubate elution fraction with open lid for 8 min at 90 C u See section 2 5 for further comments and alternative pines incubation times and temperatures for a removal of residual ethanol 20 MACHEREY NAGEL 02 2012 Rev 04 NucleoSpin Tissue XS 5 3 Protocol for paraffin embedded tissue Prepare sample As an alternative to this protocol the NucleoSpin FFPE DNA kit see ordering information is recommended for FFPE samples Prepare small sections up to 3 mg for larger samples please see the indications below from blocks of fixed embedded tissue If possible trim excess paraffin from the block before slicing Handle the sections with tweezers or toothpicks and place the samples into microcentrifuge tubes not provided Add 300 uL n octane or xylene to each tube Vortex vigorously and incubate at room temperature for about 30 min Vortex occasionally Centrifuge at 11 000 x gfor 3 min Pipette off supernatant Add 1 mL ethanol 96 100 to each tube Close and mix by inverting several times Centrifuge at 11 000 x g for 3 min Pipette off supernatant Repeat the ethanol washing step Pipette off as much of the ethanol as possible Incubate the open tube at 37 C until the ethanol has evaporated 15 min Note For samples from 3 10 mg the volumes of Buffer T1 B3 and ethanol in steps 2 3 and 4 should be doubled 160 uL each However also for larger samples the indicated volume 300
30. rough Alternative B Transfer as much as possible of the lysate solution to a 1 5 mL microcentrifuge tube not provided Discard swab and continue with recovered solution 26 MACHEREY NAGEL 02 2012 Rev 04 NucleoSpin Tissue XS Lyse sample Add one volume of Buffer B3 200 400 uL vortex 2x 5s and incubate at 70 C for 5 min Vortex briefly after the incubation Let the lysate cool down to ambient temperature Adjust binding conditions Add one volume of ethanol 96 100 200 400 uL to each sample and mix by vortexing 2x 5s Spin down briefly to clear the lid Proceed with step 5 Bind DNA of the standard protocol see section 5 1 MACHEREY NAGEL 02 2012 Rev 04 27 NucleoSpin Tissue XS 5 6 Protocol for laser microdissected tissue Before starting the preparation Check if Wash BufferB5 and Proteinase K were prepared according to section 3 Adjust thermal heating block temperature to 56 C and equilibrate sample to room temperature 18 25 C The extraction of genomic DNA from laser microdissected samples is a challenge the sample amount is very small and the DNA quality is adversely affected by fixation and staining procedures The use of cryosections or different fixation and staining procedures should always be considered as alternatives 1 Prepare sample Place laser microdisseced sample into a 1 5 mL microcentrifuge tube not provided 2 _ Pre Iyse sample
31. rtexing 2 x 5 s Spin down briefly to clear the lid Bind DNA For each sample place one NucleoSpin Tissue XS Column into a Collection Tube 2 mL Apply the sample to the column Centrifuge for 1 min at 11 000 x g Discard the flow through and place the column into a new Collection Tube 2 mL If the sample is not drawn completely through the matrix repeat the centrifugation step at 11 000 xg Wash silica membrane Add 50 uL Buffer B5 to NucleoSpin Tissue XS Column Centrifuge for 1 min at 11 000 x g It is not necessary to discard the flow through Reuse the Collection Tube Add 50 uL Buffer B5 directly onto the membrane of the NucleoSpin Tissue XS Column Centrifuge for 2 min at 11 000 x g Discard flow through with Collection Tube Elute DNA Place the NucleoSpin Tissue XS Column in a new 1 5 mL microcentrifuge tube not provided and apply 20 uL Buffer BE directly onto the center of the silica membrane of the column Centrifuge for 1 min at 11 000 x g Elution volume may be varied from approximately 5 30 uL For a correlation of elution volume DNA concentration and DNA amount eluted from the column see section 2 4 2 5 80 pL ethanol Load lysate 11 000 x g 1 min 50 pL B5 11 000 x g 1 min 50 pL B5 11 000 x g 2 min 20 uL BE 11 000 x g 1 min MACHEREY NAGEL 02 2012 Rev 04 19 NucleoSpin Tissue XS Optional Remove residual ethanol O
32. s to a silica membrane Two subsequent washing steps efficiently remove contaminations and highly pure DNA is finally eluted with 5 30 uL of a slightly alkaline elution buffer of low ionic strength 5 mM Tris HCl pH 8 5 2 2 Kit specifications NucleoSpin Tissue XS is recommended for the isolation of genomic DNA from very small samples Typical sample material comprises fresh or frozen cells tissues blood spots on Guthrie NucleoCard FTA cards see ordering information buccal swabs forensic samples and others see specifications at a glance Table 1 page 7 NucleoSpin Tissue XS is designed for high recovery of small amounts of DNA due to a special column design The special column design is connected with a significantly reduced dead volume which allows elution in as little as 5 30 uL elution buffer DNA is ready to use for downstream applications like real time PCR and others The preparation time is approximately 40 45 min for 6 12 samples exclusive incubation for lysis The DNA yield strongly depends on the sample type quality and amount see specifications at a glance Table 1 page 7 The length of the purified genomic DNA fragments depends on the quality of the sample material and may vary between 500 bp from laser microdissected or forensic samples and up to 30 kb from fresh tissues or cultured cells 6 MACHEREY NAGEL 02 2012 Rev 04 Genomic DNA from tissue Table 1 Kit specifications at a glanc
33. v 04 NucleoSpin Tissue XS 5 Protocols 5 1 Protocol for human or animal tissue Before starting the preparation Check if Wash BufferB5 and Proteinase K were prepared according to section 3 Adjust thermal heating block temperature to 56 C and equilibrate sample to room temperature 18 25 C 1 Prepare sample Place the sample of up to 2 5 mg into a 1 5 mL microcentrifuge tube not provided For samples from 2 5 10 mg double the volumes of Buffer T1 Buffer B3 and ethanol in steps 2 3 and 4 to 160 uL each 2 Pre lyse sample Add 80 uL Buffer T1 and 8 uL Proteinase K solution and mix by vortexing 2 x 5 s Be sure that the sample is 80 uL T1 completely covered with lysis solution 8 uL If processing several samples Proteinase K and Buffer T1 Proteinase K may be premixed directly before use Do never mix Buffer T1 and Proteinase K more than 10 15 min before addition to the sample Proteinase K tends to self digestion in Buffer T1 without substrate y 56 C Incubate at 56 C until complete lysis is obtained 1 4 h approximately 1 4 h or overnight Vortex occasionally during incubation or use a shaking incubator At the or end of the incubation adjust the thermal heating block temperature to 70 C for the following step 56 C overnight If RNA free DNA is crucial for downstream applications an RNase digest may be performed Add 20 uL RNase A 20 mg mL solution not included see orderin

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