Genomic DNA from tissue
Contents
1. Prepare sample Add 250 mg feces to 1 mL TE buffer 10 mM Tris Cl 1 mM EDTA pH 8 Resuspend the sample by vigorous vortexing 30 s Centrifuge the sample for 15 min at 4 000 x g Discard supernatant Resuspend the pellet in 0 2 1 mL Buffer T1 Use as much buffer as necessary for good resuspension of the sample The prepared pellet contains among other constituents cells from the digestive tract and bacteria Transfer 200 uL of the resuspended sample to a new microcentrifuge tube Proceed with the addition of 25 uL Proteinase K in step 2 of the standard protocol see section 5 Human cells bacterial cells and cells of pathogens in the stool lyse during the incubation step at 56 C with Proteinase K with different efficiency For the detection of cells that are difficult to lyse e g some bacteria and parasites it can be beneficial to perform an additional incubation at increased incubation temperature up to 95 C 5 10 min DNA yield will often be higher with such an additional incubation step at high temperature However note that the ratio of human to non human DNA will typically change due to the increased release of bacterial pathogen DNA 24 MACHEREY NAGEL 03 2014 Rev 13 NucleoSpin Tissue 6 9 Support protocol for viral DNA e g CMV from stool Before starting the preparation Check if Buffer B5 and Proteinase K were prepared according to section 3 Prepare 0 9 NaCl Set an in2. Genomic DNA from tissue User manual NucleoSpin Tissue March 2014 Rev 13 MACHEREY NAGEL www mn net com SUN Genomic DNA from tissue Protocol at a glance Rev 13 1 Prepare sample NucleoSpin Tissue Cut 25 mg into small pieces 2 Pre lyse sample 180 uL T1 25 uL Proteinase K 56 C 1 3 h 3 Lyse sample 200 uL B3 70 C 10 min 4 Adjust DNA binding conditions 210 uL 96 100 ethanol 5 Bind DNA Load all 11 000 x g 1 min 6 Wash silica membrane 1 and 2 4 eS 1 wash 2rd wash 500 uL BW 600 uL B5 11 000 x g 1 min 7 Dry silica membrane S 11 000 x g 1 min 8 Elute highly pure DNA 100 uL BE 70 C RT 1 min 11 000 x g 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 tissue Table of contents 1 Components 5 1 1 Kit contents 5 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 The basic principle 7 2 2 Kit specifications 7 2 3 Elution procedures 8 3 Storage conditions and preparation of working solutions 10 4 Safety instructions risk and safety phrases 11 4 1 Risk and safety phrases 11 4 2 GHS classification 12 5 Standard protocol for human o
3. 30 31 33 34 35 37 37 39 40 MACHEREY NAGEL 03 2014 Rev 13 Genomic DNA from tissue 1 Components 1 1 Kit contents NucleoSpin Tissue 10 preps 50 preps 250 preps REF 740952 10 740952 50 740952 250 Lysis Buffer T1 5 mL 20 mL 100 mL Lysis Buffer B3 7 5 mL 15 mL 75 mL Wash Buffer BW 6 mL 30 mL 2x75 mL Wash Buffer B5 4mL 2x7mL 2 x 40 mL Concentrate Elution Buffer BE 3 mL 15 mL 75 mL Proteinase K 6 mg 30 mg 2x75 mg lyophilized Proteinase Buffer PB 0 8 mL 1 8 mL 8 mL NucleoSpin Tissue 10 50 250 Columns light green rings Collection Tubes 2 mL 20 100 500 User manual 1 1 1 Composition of Elution Buffer BE 5 mM Tris HCl pH 8 5 For preparation of working solutions and storage see section 3 MACHEREY NAGEL 03 2014 Rev 13 5 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 Heating block for incubation at 70 C Equipment for sample disruption and homogenization 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 kit is used for the first time Experienced users however may
4. 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 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 off
5. or use a shaking water bath Samples can be incubated overnight as well 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 ordering information and incubate for an additional 5 min at room temperature Proceed with step 3 of the standard protocol see section 5 Other protocols use 5 200 U lyticase or zymolase depending on enzyme quality or brand Increasing the enzyme concentration may be required if spheroplasts are not formed MACHEREY NAGEL 03 2014 Rev 13 19 NucleoSpin Tissue 6 4 Support protocol for dried blood spots e g NucleoCards FTA cards Guthrie cards Before starting the preparation Check if Buffer B5 and Proteinase K were prepared according to section 3 Set an incubator or water bath to 56 C Before elution preheat Elution Buffer BE to 70 C Prepare sample Cut out one or two dried blood spots as accurately as possible Cut spots into small pieces and place them in a 1 5 mL microcentrifuge tube not provided The area of the dried blood spots should be between 15 and 30 mm Pre lyse sample Add 180 uL Buffer T1 and mix by vortexing Place the samples in a water bath or heating block and heat for 10 min at 94 C Let the sample cool down Add 25 uL Proteinase K solution Spin the samples briefly vortex and incubate at 56 C for 1 h Vortex occasionally during incubation o
6. protocol for mouse or rat tails Before starting the preparation Check if Buffer B5 and Proteinase K were prepared according to section 3 Set an incubator or water bath to 56 C Before elution preheat Elution Buffer BE to 70 C 1 Prepare sample Cut two 0 6 cm pieces of mouse tail and place them in a 1 5 mL centrifuge tube not provided If processing rat tails one 0 6 cm piece is sufficient 2 Pre lyse sample Add 180 uL Buffer T1 and 25 uL Proteinase K and vortex Incubate at 56 C overnight or until complete lysis is obtained Lysis time can substantially be reduced down to approximately one hour if the tissue is broken up mechanically e g if the tissue is cut into very small pieces before lysis Vortex occasionally during incubation or use a shaking water bath To remove residual bones or hair centrifuge for 5 min at high speed e g 11 000 x g Transfer 200 pL supernatant to a new tube 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 200 uL Buffer B3 to the lysate and vortex vigorously Buffer B3 and ethanol see step 4 can be premixed before addition to the lysate 4 Adjust DNA binding conditions Add 210 uL ethanol to the lysate and vortex vigorously Proceed with s
7. the columns DNA yield 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 done with buffers with a pH lt 7 0 Use slightly alkaline elution buffers like Buffer BE pH 8 5 Especially when expecting high yields from large amounts of material we recommend elution with 200 uL Buffer BE and incubation of the closed columns in an incubator at 70 C for 5 min before centrifugation Incomplete lysis Sample not thoroughly homogenized and mixed with Buffer T1 Poor DNA Proteinase K The mixture has to be vortexed vigorously quality immediately after the addition of Buffer T1 Decreased Proteinase K activity Store dissolved Proteinase K at 20 C for 6 months MACHEREY NAGEL 03 2014 Rev 13 37 Genomic DNA from tissue Reagents not applied properly Prepare Buffer B5 and Proteinase K solution according to instructions see section 3 Add ethanol to the lysates before Poor DNA loading them on the columns quality continued RNA in sample e If RNA free DNA is desired add 20 uL of RNase A solution 10 mg mL not supplied with the kit before addition of Buffer B3 and incubate at 37 C for 5 min Too much sample material used Do not use more sample material than recommended 25 mg for most tissue types If insoluble material like bones or ha
8. 3 Check if PBS is available Set an incubator or water bath to 56 C Before elution preheat Elution Buffer BE to 70 C Prepare sample Collect the samples with cotton dacron Daigger or C E P swabs Gibco BRL Scrape firmly against the inside of 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 sample 2a Pre lyse sample Place the dry swab material in 2 mL microcentrifuge tubes not provided Add 400 600 uL PBS and 25 uL Proteinase K solution to the swabs The volume of PBS is depending on the type of swab used for cotton and dacron swabs 400 uL are sufficient for C E P swabs 600 uL are necessary Mix by vortexing 2 x 5 s and incubate 10 min at 56 C Separate lysis solution from buccal swabs Alternative A Place a NucleoSpin Filter not provided see ordering information into a Collection Tube 2mL 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 through 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 MACHEREY NAGEL 03 2014 Rev 13 35 NucleoSpin Tissue 3 Lyse sample Add one volume Buffer B3 400 or 600 uL
9. 60 uL Add 1 35 mL Add 3 35 mL Proteinase Buffer Proteinase Buffer Proteinase Buffer to each vial 10 MACHEREY NAGEL 03 2014 Rev 13 Genomic DNA from tissue 4 Safety instructions risk and safety phrases The following components ofthe NucleoSpin Tissue 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 36 50 x Xn R 22 36 S 26 39 Guanidinhydrochlorid 36 50 BW Guanidine hydrochloride 36 50 x Xn R 10 22 S 16 26 isopropanol 20 50 36 67 39 Guanidinhydrochlorid 36 50 Isopropanol 20 50 Proteinase K Proteinase K lyophilized x Xn R 36 37 38 S 22 24 Proteinase K lyophilisiert 42 26 36 37 Risk phrases R10 Flammable Entztindlich R 22 Harmful if swallowed Gesundheitssch dlich beim Verschlucken R 36 Irritating to eyes Reizt die Augen R 36 37 38 Irritating to eyes respiratory system and skin Reizt die Augen Atmungsorgane und die Haut R 42 May cause sensitization by inhalation Sensibilisierung durch Einatmen m glich R 67 Vapours may cause drowsiness and dizziness D mpfe k nnen Schl frigkeit und Benommenheit verursachen Safety phrases S16 Keep away from sources of ignition No Smoking Von Z ndquellen fernha
10. L 03 2014 Rev 13 9 Genomic DNA from tissue 3 Storage conditions and preparation of working solutions Attention Buffers B3 and BW contain chaotropic salt Wear gloves and goggles CAUTION Buffers B3 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 for at least one year During storage especially at low temperatures a white precipitate may form in Buffer T1 or B3 Such precipitates can be easily dissolved by incubating the bottle at 50 70 C before use Before starting any NucleoSpin Tissue protocol prepare the following Wash Buffer B5 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 Wash Buffer B5 can be stored at room temperature 18 25 C for at least 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 at least 6 months NucleoSpin Tissue 10 preps 50 preps 250 preps REF 740952 10 740952 50 740952 250 Wash 4mL 2x7mL 2x 40 mL Buffer B5 Add 16 mL ethanol Add 28 mL ethanol Add 160 mL ethanol Concentrate to each bottle to each bottle Proteinase K 6 mg 30 mg 2x75 mg Add 2
11. anol 319 336 3014312 20 50 D 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 334 335 304 340 305 351 338 312 332 313 337 313 342 311 403 233 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 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 verursa chen H 335 May cause respiratory irritation Kann die Atemwege reizen H 336 May cause drowsiness or dizziness Kann Schl frigkit und Benommenheit verursachen 12 MACHEREY NAGEL 03 2014 Rev 13 Genomic DNA from tissue 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 sparks open flames hot surfaces No smoking Von Hitze Funken offener Flamme heiBen Oberfl chen fernhalten Nicht rauchen Keep container tightly closed Beh lter dicht verschlossen halten Avoid breathing dust Einatm
12. aphin and incubate for 30 60 min at 37 C Add 25 uL Proteinase K incubate at 56 C until complete lysis is obtained Proceed with step 3 of the standard protocol see section 5 18 MACHEREY NAGEL 03 2014 Rev 13 NucleoSpin Tissue 6 3 Support protocol for yeast Before starting the preparation Check if Buffer B5 and Proteinase K were prepared according to section 3 Check that sorbitol buffer and Iyticase or zymolase not provided with the kit is available for sample pre lysis Set an incubator or water bath to 30 C and 56 C Before elution preheat Elution Buffer BE to 70 C Prepare sample Harvest 3 mL YPD yeast culture ODs lt 10 by centrifugation for 10 min at 5 000 x g Wash the cells once with 1 mL 10 mM EDTA pH 8 Remove the supernatant and pellet the cells by centrifugation 5 000 x g 10 min Pre lyse sample Resuspend the pellet in 600 uL sorbitol buffer 1 2 M sorbitol 10 mM CaCl 0 1M Tris HCl pH 7 5 35mM B mercaptoethanol Add 50 U Iyticase or zymolase Incubate at 30 C for 30 min This step degrades the yeast cell wall creating spheroplasts Spheroplast formation may be checked microscopically Centrifuge the mixture for 10 min at 2 000 x gremove supernatant and resuspend the pelleted spheroplasts in 180 uL Buffer T1 Add 25 uL Proteinase K solution and vortex vigorously Incubate at 56 C until complete lysis is obtained at least 1 3 h Vortex occasionally during incubation
13. blood The purified DNA can be used directly for PCR Southern blotting or any kind of enzymatic reactions The kit allows purification of up to 35 ug of pure genomic DNA with an A260 A280 ratio between 1 7 and 1 9 The NucleoSpin Tissue Column is capable of binding up to 60 ug of genomic DNA For lysis of certain bacterial and yeast strains additional enzymes may be necessary which are not part of this kit See the relevant support protocol for details Table 1 Kit specifications at a glance Parameter NucleoSpin Tissue Technology Silica membrane technology Format Mini spin column lt 25 mg tissue Sample material 10 107 cultured cells Typical yield 20 35 ug Elution volume 60 100 uL Preparation time 20 min prep excluding Iysis Binding capacity 60 ug MACHEREY NAGEL 03 2014 Rev 13 7 Genomic DNA from tissue Forensic quality product NucleoSpin Tissue is certified as forensic quality product Consumables used in forensics need to be treated carefully to prevent DNA contamination MACHEREY NAGEL therefore has a stringently controlled production process to avoid DNA contamination of consumables Further MACHEREY NAGEL uses ethylene oxide EO treatment to remove amplifiable DNA which might still be introduced during the manufacturing process MACHEREY NAGEL products carrying the forensic quality seal contain plastic materials that are EO treated This means DNA of any kind which might sti
14. cant supernatant If frozen urine samples are used precipitates may appear after defrosting which must be dissolved before the centrifugation step This can be done through a 30 min incubation step at 37 40 C If a complete solution does not happen let the precipitate sediment and proceed with step 1 of the support protocol using only the supernatant 2 Pre lyse sample Resuspend the pellet in 180 pL Buffer T1 and 25 uL Proteinase K Resuspend the first pellet in 180 uL Buffer T1 and 25 uL Proteinase K Transfer the resuspended solution of the first tube to the second tube and the resuspended solution of the second tube to the third tube and so on Finally continue with step 3 3 Lyse sample Add 200 uL Buffer B3 vortex and incubate at least for 20 min at 70 C 4 Adjust DNA binding conditions Add 210 uL ethanol 96 100 to the sample and vortex vigorously 5 Bind DNA For each sample place one NucleoSpin Tissue Column into a Collection Tube Apply the sample to the column Centrifuge for 1 min at 4 500 x g Discard the flow through and place the column back into the Collection Tube MACHEREY NAGEL 03 2014 Rev 13 31 NucleoSpin Tissue 6 Wash silica membrane Add 500 uL Buffer BW Centrifuge for 1 min at 4 500 x g Discard flow through and place the column back into the Collection Tube Add 600 uL Buffer B5 to the column and centrifuge for 2 min at 11 000 x g Discard flow through and p
15. 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 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 40 MACHEREY NAGEL 03 2014 Rev 13 Genomic DNA from tissue components not manufactured by MACHEREY
16. cubator or water bath to 56 C Before elution preheat Elution Buffer BE to 70 C Prepare sample Suspend the stool sample in 0 9 NaCl ca 0 5 g in max 4 mL Centrifuge aliquots of the stool sample for 5 min at 800 x g at RT e g 4 mL 4x 1mLina 1 5 mL microcentrifuge tube Carefully reunite supernatant do not touch the pellet Filtrate supernatant through 0 22 0 45 um sterile filter Fractionate the filtrate and centrifuge for 1 min at 11 000 x g Pre lyse sample Carefully remove the supernatant by decanting Add 400 uL Buffer T1 and 35 uL Proteinase K and mix by vortexing Lyse sample Add 400 pL Buffer B3 and mix by vortexing Incubate for at least 30 min at 70 C Adjust DNA binding conditions Add 420 uL ethanol 96 100 and mix by vortexing Bind DNA For each sample place one NucleoSpin Tissue Column into a Collection Tube Load the NucleoSpin Tissue Column successively Centrifuge for 1 min at 4 500 x g Discard the flow through and place the column back into the Collection Tube If the sample is not drawn completely through the matrix repeat the centrifugation step at 11 000 x g Discard flow through MACHEREY NAGEL 03 2014 Rev 13 25 NucleoSpin Tissue 6 Wash silica membrane Add 600 uL Buffer BW Centrifuge for 1 min at 4 500 x g Discard flow through and place the column back into the Collection Tube Add 600 uL Buffer B5 to the column and c
17. dard protocol see section 5 MACHEREY NAGEL 03 2014 Rev 13 29 NucleoSpin Tissue 6 13 Support protocol for purification of bacterial DNA e g Borrelia burgdorferi from urine Before starting the preparation Check if Buffer B5 and Proteinase K were prepared according to section 3 Set an incubator or water bath to 56 C Before elution preheat Elution Buffer BE to 70 C Prepare sample Centrifuge 1 mL urine sample at 13 000 x g for 30 min Discard supernatant add again 1 mL urine sample to the pellet and centrifuge at 13 000 x g for 30 min This step a can be repeated up to three times The sample material should be fresh and stored at 20 C to 80 C is only recommended for a couple of days After thawing incubate the sample at 40 C as long as all precipitates are dissolved Urine tends to form precipitates when stored at low temperatures Proceed with step 2 of the standard protocol see section 5 30 MACHEREY NAGEL 03 2014 Rev 13 NucleoSpin Tissue 6 14 Support protocol for purification of viral DNA e g CMV from urine Before starting the preparation Check if Buffer B5 and Proteinase K were prepared according to section 3 Set an incubator or water bath to 56 C Before elution preheat Elution Buffer BE to 70 C 1 Prepare sample Centrifuge aliquots of the urine sample for 10 min at full speed e g 4 mL 4x1 mLina 1 5 mL microcentrifuge tube Carefully de
18. depending on the swab type volume of PBS buffer used and vortex vigorously Incubate the samples at 70 C for 10 min Note Depending on the number of preparations additional Buffer B3 might be needed see ordering information 4 Adjust DNA binding conditions Add one volume 96 100 ethanol 400 or 600 uL depending on the swab type to each sample and mix by vortexing 5 Bind DNA Transfer 600 uL of the samples from the 2 mL microcentrifuge tubes into NucleoSpin Tissue Columns Centrifuge at 11 000 x g for 1min If the samples are not drawn through completely repeat the centrifugation Discard flow through Place the columns back into the Collection Tubes and repeat step 5 once or twice depending on the lysis volume When all of the lysate has been applied to the columns proceed with step 6 of the standard protocol section 5 36 MACHEREY NAGEL 03 2014 Rev 13 Genomic DNA from tissue 7 Appendix 7 1 Troubleshooting Problem Possible cause and suggestions Incomplete Iysis Sample not thoroughly homogenized and mixed with Buffer T1 Proteinase K The mixture has to be vortexed vigorously immediately after the addition of Buffer T1 Decreased Proteinase K activity Store dissolved Proteinase K at 20 C for 6 months Reagents not applied properly Prepare BufferB5 and Proteinase K solution according to instructions see section 3 Add ethanol to the lysates before No or poor loading them onto
19. en von Staub vermeiden Wear protective gloves eye protection Schutzhandschuhe Augenschutz tragen IF SWALLOWED Call a POISON CENTER or doctor physician if you feel unwell BEI VERSCHLUCKEN Bei Unwohlsein GIFTINFORMATIONSZENTRUM oder Arzt anrufen IFON SKIN Wash with plenty of soap and 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 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 GIFTINGORMATIONSZENTRUM oder 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 anhaltender Augenreizung Arztlichen Rat einholen rztliche Hilfe hinzuziehen If experiencing respiratory symptoms Call a POISON CENTER or doc tor physician Bei Symptomen der Atemwege Giftinformationszentrum oder Arzt anrufen Store in a well ventilated place Keep container tightly c
20. ent Regarding these products or services we can not grant any guarantees regarding selection efficiency or operation MACHEREY NAGEL 03 2014 Rev 13 41
21. entrifuge for 1 min at 4 500 x g Discard flow through and place the column back into the Collection Tube Add 600 uL Buffer B5 to the column and centrifuge for 2 min at 11 000 x g Discard flow through 7 Dry silica membrane Place the NucleoSpin Tissue Column into a new Collection Tube and incubate with open lid for 1 2 min at 70 C Residual ethanol is removed during this step 8 Elute highly pure DNA Place the NucleoSpin Tissue Column into a 1 5 mL microcentrifuge tube not provided and add 100 pL prewarmed Buffer BE 70 C Incubate with closed lid for 3 5 min at 70 C Centrifuge for 1 min at 4 500 x g For alternative elution procedures see section 2 3 Use 10 uL DNA extract for a 20 uL PCR reaction mix Add inhibition control mix 10 uL DNA extract with human DNA and amplify with for example actin B globin or other human specific primer 26 MACHEREY NAGEL 03 2014 Rev 13 NucleoSpin Tissue 6 10 Support protocol for detection of Mycobacterium tuberculosis or Legionella pneumophila in sputum or bronchoalveolar lavage Before starting the preparation Check if Buffer B5 and Proteinase K were prepared according to section 3 Set an incubator or water bath to 56 C Before elution preheat Elution Buffer BE to 70 C Prepare N acetyl cystein Na0H 2g NaOH 1 45g sodium citrate 0 5 g N acetyl cystein Add water to 100 mL 1 Prepare sample Add 200 500 uL sputum or bronchoalveolar lavage to an equal vol
22. hibit enzymatic reactions Repurify DNA and elute in Buffer BE If the Asgo Azgo ratio of the eluate is below 1 6 repeat the purification procedure Add 1 volume Buffer B3 plus 1 volume ethanol 96 100 to the eluate Load the mixture onto a NucleoSpin Tissue Column and proceed with step 5 of the 7 2 Ordering information Product REF Pack of NucleoSpin Tissue NucleoSpin Tissue XS NucleoSpin Blood NucleoSpin FFPE DNA Buffer T1 Buffer B3 Buffer B5 Concentrate for 100 mL Buffer B5 Buffer BW Proteinase K RNase A NucleoSpin Forensic Filters NucleoSpin Forensic Filters Bulk NucleoSpin DNA Trace Collection Tubes 2 mL NucleoSpin Filters NucleoCard 740952 10 50 250 740901 10 50 250 740951 10 50 250 740980 10 50 250 740940 25 740920 740921 740922 740506 740505 50 740505 740988 10 50 250 10 50 250 preps 10 50 250 preps 10 50 250 preps 10 50 250 preps 25 mL 100 mL 20 mL 100 mL 100 mg 50 mg 10 50 250 pieces 740988 50B 250B 1000B 50 250 1000 pieces 740942 4 25 740600 740606 740403 10 100 Visit www mn net com for more detailed product information 4 25 preps 1000 50 10 100 cards MACHEREY NAGEL 03 2014 Rev 13 39 Genomic DNA from tissue 7 3 Product use restriction warranty NucleoSpin Tissue kit components are intended developed designed and sold FOR RESEARCH PURPOSES ONLY e
23. icer 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 E I du Pont de Nemours and Company FTA is a registered trademark of Whatman Ltd Polytron is a registered trademark of Dragerwerk AG Ultra Thurrax is a registered trademark of IKA Works Inc NucleoSpin is a registered 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 of fend against trademarks and brands and can not be seen as a kind of recommendation or assessm
24. in 2 mL sterile water Centrifuge for 10 min at 10 000 x g Pre lyse sample Resuspend the pellet in 180 uL Buffer T1 and add 25 uL Proteinase K solution Carry out the standard protocol beginning with step 3 see section 5 After elution of the DNA proceed with the following step Precipitate the DNA by adding 20 pL 3 2 M sodium acetate and 400 uL ethanol to 200 uL eluate Centrifuge for 30 min at 11 000 x g Discard supernatant and wash the pellet with 1 mL 70 ethanol and resuspend in 10 uL sterile water 28 MACHEREY NAGEL 03 2014 Rev 13 NucleoSpin Tissue 6 12 Support protocol for purification of bacterial DNA e g Chlamydia trachomatis from cultures biological fluids or clinical specimens Before starting the preparation Check if Buffer B5 and Proteinase K were prepared according to section 3 Set an incubator or water bath to 56 C Before elution preheat Elution Buffer BE to 70 C 1 Prepare sample Isolation of bacterial DNA from bacterial cultures or biological fluids Pellet bacteria by centrifugation for 5 min at 13 000 x g and proceed with step 2 of the standard protocol see section 5 e Isolation of bacterial DNA from eye nasal or pharyngeal swabs Collect samples add 2 mL PBS containing a common fungicide and incubate for several hours at room temperature Pellet bacteria by centrifugation for 5 min at 13 000 x g Discard supernatant Proceed with step 2 of the stan
25. inase K to each sample Close the microcentrifuge tube and spin briefly for 15 s at 1 500 x g in order to get the swab material completely submerged Incubate at room temperature for 5 min Vortex the tube vigorously for 15 s and spin briefly for 15 s at 1 500 x g Incubate the tubes at 70 C in an incubator for 10 min Place a weight on top of the tube in order to prevent the caps from popping off Shift the temperature to 95 C for 5 min Spin briefly for 15 s at 1 500 x g to collect any sample from the lids Open the microcentrifuge tubes Skip incubation at 95 C depending on the detecting bacterial strain 2a Separate lysis solution from dental swabs Alternative A Place a NucleoSpin Filter not provided see ordering information into a Collection Tube 2mL 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 through 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 Proceed with step 3 of the standard protocol see section 5 34 MACHEREY NAGEL 03 2014 Rev 13 NucleoSpin Tissue 6 17 Support protocol for purification of genomic DNA from buccal swabs Before starting the preparation Check if Buffer B5 and Proteinase K were prepared according to section
26. ir remains in the lysate spin down the debris and transfer the clear supernatant to a new microcentrifuge tube before proceeding with addition of Buffer B3 and ethanol The use of the NucleoSpin Filter prior column loading prevents column clogging Clogged Incomplete lysis columns Sample not thoroughly homogenized and mixed with Buffer T1 Proteinase K The mixture has to be vortexed vigorously immediately after the addition of Buffer T1 Decreased Proteinase K activity Store dissolved Proteinase K at 20 C for 6 months Reagents not applied properly e Prepare Buffer B5 and Proteinase K solution according to instructions see section 3 Add ethanol to the lysates before loading them on the columns Carry over of ethanol or salt Suboptimal Make sure to centrifuge 1 min at 11 000 x gin order to remove performance all of ethanolic Buffer 5 before eluting the DNA If for any of genomic reason the level of Buffer B5 has reached the column outlet DNA in after drying repeat the centrifugation enzymatic Do not chill Buffer B5 before use Cold buffer will not remove reactions salt effectively Equilibrate Buffer B5 to room temperature before use 38 MACHEREY NAGEL 03 2014 Rev 13 Genomic DNA from tissue Suboptimal performance of genomic DNA in s enzymatic reactions continued standard protocol see section 5 Contamination of DNA with inhibitory substances Do not elute DNA with TE buffer EDTA may in
27. l 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 MACHEREY NAGEL 03 2014 Rev 13 Genomic DNA from tissue Convenient elution For convenience elution buffer of ambient temperature may be used This will result in a somewhat lower yield approximately 20 compared to elution with heated 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 temperature by inhibition of omnipresent DNases However EDTA interferes depending on the final concentration with certain downstream applications Note Elution Buffer BE 5 mM Tris HCl pH 8 5 provided with the kit does not contain EDTA For optimal performance of isolated DNA in downstream applications we recommend eluting with the supplied elution buffer and storage especially long term at 20 C Freeze thaw cycles will have no effect on most downstream applications Possible exceptions are detection of trace amounts of DNA or long range PCR e g gt 10 kbp Multiple freeze thaw cycles or storing DNA at 4 C or room temperature may influence detection sensitivities or reaction efficiencies due to DNA shearing or adsorption to surfaces MACHEREY NAGE
28. lace the column back into the Collection Tube Dry silica membrane Incubate with open lid for 1 2 min at 70 C Residual ethanol is removed during this step Elute highly pure DNA Add 70 uL prewarmed Buffer BE 70 C close the lid and incubate for further 3 5 min at 70 C Centrifuge for 1 min at 4 500 x g 32 MACHEREY NAGEL 03 2014 Rev 13 NucleoSpin Tissue 6 15 Support protocol for purification of genomic DNA from insects Before starting the preparation Check if Buffer B5 and Proteinase K were prepared according to section 3 Set an incubator or water bath to 56 C Before elution preheat Elution Buffer BE to 70 C 1 Prepare sample Homogenize not more than 50 mg insects under liquid nitrogen and transfer the powder into a 1 5 mL microcentrifuge tube not provided Proceed with step 2 of the standard protocol see section 5 with addition of Proteinase K MACHEREY NAGEL 03 2014 Rev 13 33 NucleoSpin Tissue 6 16 Support protocol for purification of genomic DNA from dental swabs Before starting the preparation Check if Buffer B5 and Proteinase K were prepared according to section 3 Set an incubator or water bath to 56 C Before elution preheat Elution Buffer BE to 70 C 1 Prepare sample Place swab material paper cotton brushes plastic in a 1 5 mL microcentrifuge tube not provided 2 Pre lyse sample Add 180 uL Buffer T1 and 25 uL Prote
29. ll be introduced into plastic consumables during the production process is inactivated by means of the treatment with ethylene oxide in order to prevent the generation of accidental human profile by PCR amplification Ethylene oxide treatment has been shown to be the method of choice to prevent DNA profiles due to DNA contamination Shaw et al 2008 Figure 1 UV Gamma Electron beam Ethylene oxide 100 30 40 27 13 87 Nu gt N 30 70 Full profile Partial profile loadable E Partial profile unloadable HB No profile Figure 1 According to Shaw et al 2008 Comparison of the effects of sterilization techniques on 2 3 subsequent DNA profiling Int J Legal Med 122 29 33 Elution procedures In addition to the standard method recovery rate about 70 90 several modifications are possible to increase yield concentration and convenience 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 approximately 30 higher than with standard elution The yield of eluted nucleic acid will be about 80 High yield and high concentration Apply half the volume of elution buffer as indicated in the individua
30. losed Beh lter dicht geschlossen 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 03 2014 Rev 13 13 NucleoSpin Tissue 5 Standard protocol for human or animal tissue and cultured cells Before starting the preparation Check if Buffer B5 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 1 Prepare sample Cut 25 mg human or animal tissue into small pieces Place the sample in a microcentrifuge tube not provided Proceed with step 2 Samples that are difficult to lyse can be ground under liquid nitrogen or may be treated in a mechanical homogenizer Polytror Ultra Turrax Add 25 mg of tissue to a 1 5 mL microcentrifuge tube not provided add 50 75 uL phosphate buffered saline PBS and homogenize Cultured cells Resuspend up to 10 cells in a final volume of 200 pL Buffer T1 Add 25 pL Proteinase K solution and 200 pL Buffer B3 Incubate the sample at 70 C for 10 15 min Proceed with step 4 2 Pre lyse sample Add 180 uL Buffer T1 and 25 uL Proteinase K solution 180 pL T1 Vortex to mix Be sure that the samples are completely covered wi
31. lten Nicht rauchen S22 Do not breathe dust Staub nicht einatmen S24 Avoid contact with the 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 Hazard labeling not necessary if quantity per bottle below 125g or ml certificate of exemption according to 67 548 EEC Art 25 1999 45 EC Art 12 and German GefStoffV 20 3 and TRGS 200 7 1 For further information see Material Safety Data Sheet MACHEREY NAGEL 03 2014 Rev 13 11 Genomic DNA from tissue Safety phrases 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 39 Wear suitable eye face protection Schutzbrille Gesichtsschutz tragen 4 2 GHS classification Only harmful features do not 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 eJ ats Hazard Precaution symbol 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 BW Guanidine hydrochloride amp Warning 226 302 210 233 280 36 50 isoprop
32. ly the sample to the column Centrifuge for 1 min at 11 000 x g Discard the flow through and place the column back into the Collection Tube If the sample is not drawn completely through the matrix repeat the centrifugation step at 11 000 xg Discard flow through 56 C 1 3h pon 56 C overnight 200 uL B3 70 C 10 min e 210 uL ethanol i Vortex Load samples 11 000 x g 1 min MACHEREY NAGEL 03 2014 Rev 13 15 NucleoSpin Tissue 6 Wash silica membrane 500 pL BW 1 wash Ss 11 000 x g Add 500 uL Buffer BW Centrifuge for 1min at 1 min 11 000 x g Discard flow through and place the column i back into the Collection Tube 2 wash amp Smee Add 600 pL Buffer B5 to the column and centrifuge for 11 000 x g 1 min at 11 000 x g Discard flow through and place the 1 min column back into the Collection Tube 6 Dry silica membrane Centrifuge the column for 1 min at 11 000 x g 1 Pie 9 Residual ethanol is removed during this step 7 Elute highly pure DNA 100 uL BE 70 C Place the NucleoSpin Tissue Column into a 1 5 mL microcentrifuge tube not provided and add 100 pL RT prewarmed Buffer BE 70 C Incubate at room 1 min temperature for 1 min Centrifuge 1 min at 11 000 x g es 11 000 x g For alternative elution procedures see section 2 3 1 min 16 MACHEREY NAGEL 03 2014 Rev 13 NucleoSpin Tissue 6 Support protocols 6 1 Support
33. r animal tissue and cultured cells 14 6 Support protocols 17 6 1 Support protocol for mouse or rat tails 17 6 2 Support protocol for bacteria 18 6 3 Support protocol for yeast 19 6 4 Support protocol for dried blood spots e g NucleoCards FTA cards Guthrie cards 20 6 5 Support protocol for genomic DNA and viral DNA from blood samples 21 6 6 Support protocol for hair roots 22 6 7 Support protocol for paraffin embedded tissue 23 6 8 Support protocol for genomic DNA from stool 24 6 9 Support protocol for viral DNA e g CMV from stool 25 6 10 Support protocol for detection of Mycobacterium tuberculosis or Legionella pneumophila in sputum or bronchoalveolar lavage 27 6 11 Support protocol for detection of EHEC bacteria in food e g fresh cows milk 28 MACHEREY NAGEL 03 2014 Rev 13 3 Genomic DNA from tissue 6 12 Support protocol for purification of bacterial DNA e g Chlamydia trachomatis from cultures biological fluids or clinical specimens 6 13 Support protocol for purification of bacterial DNA e g Borrelia urgdorferi from urine 6 14 Support protocol for purification of viral DNA e g CMV from urine 6 15 Support protocol for purification of genomic DNA from insects 6 16 Support protocol for purification of genomic DNA from dental swabs 6 17 Support protocol for purification of genomic DNA from buccal swabs Appendix 7 1 Troubleshooting 7 2 Ordering information 7 3 Product use restriction warranty 29
34. r or clotted blood samples 4 Adjust DNA binding conditions Add 210 uL ethanol 96 100 to each sample and vortex again 5 Bind DNA For each preparation take one NucleoSpin Tissue 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 Proceed with step 6 Wash silica membrane of the standard protocol see section 5 MACHEREY NAGEL 03 2014 Rev 13 21 NucleoSpin Tissue 6 6 Support protocol for hair roots Before starting the preparation Check if Buffer B5 and Proteinase K were prepared according to section 3 Set an incubator or water bath to 56 C Before elution preheat Elution Buffer BE to 70 C 1 Prepare sample Cut off the hair roots from the hair sample up to 100 and collect them in a 1 5 mL microcentrifuge tube not provided 2 Pre lyse sample Add 180 uL Buffer T1 to the hair roots and freeze the samples in liquid nitrogen Thaw samples in a 56 C water bath Repeat this procedure 4 times Add 25 uL Proteinase K solution mix by vortexing and incubate 6 8 h or overnight at 56 C Use a shaking water bath or vortex occasionally Proceed with step 3 of the standard protocol see section 5 22 MACHEREY NAGEL 03 2014 Rev 13 NucleoSpin Tissue 6 7 Support protocol fo
35. r paraffin embedded tissue Before starting the preparation Check if Buffer B5 and Proteinase K were prepared according to section 3 Check if xylene or n octane is available Set an incubator or water bath to 37 C and 56 C Before elution preheat Elution Buffer BE to 70 C Prepare sample Prepare small sections up to 25 mg 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 Add 1 mL 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 g for 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 Proceed with step 2 of the standard protocol see section 5 MACHEREY NAGEL 03 2014 Rev 13 23 NucleoSpin Tissue 6 8 Support protocol for genomic DNA from stool Before starting the preparation Check if Buffer B5 and Proteinase K were prepared according to section 3 Check if TE buffer is available Set an incubator or water bath to 56 C Before elution preheat Elution Buffer BE to 70 C
36. r use a shaking water bath Make sure that the samples are completely covered with lysis buffer during incubation Lyse sample Add 200 uL Buffer B3 vortex vigorously to mix and incubate at 56 C for 10 min Proceed with step 4 of the standard protocol see section 5 20 MACHEREY NAGEL 03 2014 Rev 13 NucleoSpin Tissue 6 5 Support protocol for genomic DNA and viral DNA from blood samples Before starting the preparation Check if Buffer B5 and Proteinase K were prepared according to section 3 Before elution preheat Elution Buffer BE to 70 C 1 2 Prepare sample Pre lyse sample Not necessary 3 Lyse blood sample Pipette 25 uL Proteinase K and up to 200 uL blood buffy coat or body fluid sample equilibrated to room temperature into 1 5 mL microcentrifuge tubes not provided For sample volumes less than 200 uL add PBS to adjust the volume to 200 uL If purifying DNA viruses we recommend starting with 200 uL serum or plasma If cultured cells are used resuspend up to 5 x 10 cells in a final volume of 200 uL PBS Add 200 uL Buffer B3 to the samples and vortex the mixture vigorously 10 20 s Incubate samples at room temperature for 5 min Mix 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 olde
37. 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 Note Buffer B3 is delivered premixed now 6 MACHEREY NAGEL 03 2014 Rev 13 Genomic DNA from tissue 2 Product description 2 1 The basic principle With the NucleoSpin Tissue method genomic DNA can be prepared from tissue cells e g bacteria and many other sources Lysis is achieved by incubation of the sample material in a proteinase K SDS solution Appropriate conditions for DNA binding to the silica membrane in the NucleoSpin Tissue Columns are achieved by the addition of chaotropic salts and ethanol to the lysate The binding process is reversible and specific to nucleic acids Contaminations are removed by subsequent washing with two different buffers Pure genomic DNA is finally eluted under low ionic strength conditions in a slightly alkaline elution buffer 2 2 Kit specifications NucleoSpin Tissue is designed for the rapid small scale preparation of highly pure genomic DNA from any tissue cells bacteria yeast forensic samples serum plasma or other body fluids It is also suitable for preparation of DNA from human or animal
38. tep 5 of the standard protocol see section 5 MACHEREY NAGEL 03 2014 Rev 13 17 NucleoSpin Tissue 6 2 Support protocol for bacteria Before starting the preparation Check if Buffer B5 and Proteinase K were prepared according to section 3 Set an incubator or water bath to 56 C Before elution preheat Elution Buffer BE to 70 C 1 Prepare sample Up to 1 mL of bacterial culture can be used for the preparation depending on for example density of culture culture medium and bacterial strain Centrifuge up to 1 mL culture for 5 min at 8 000 x g Remove supernatant 2 Pre lyse sample Resuspend the pellet in 180 uL Buffer T1 by pipetting up and down Add 25 pL Proteinase K Vortex vigorously and incubate at 56 C until complete lysis is obtained at least 1 3 h Vortex occasionally during incubation or use a shaking incubator Samples can be incubated overnight as well 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 ordering information and incubate for an additional 5 min at room temperature Hard to lyse bacteria Some strains especially Gram positive bacteria are more difficult to lyse In such cases a preincubation with a lytic enzyme is necessary Resuspend the pelleted cells in 20 mM Tris HCl 2 mM EDTA 1 Triton X 100 pH 8 instead of Buffer T1 supplemented with 20 mg mL lysozyme or 0 2 mg mL lysost
39. th lysis solution U 25 pL f Proteinase K If processing several samples Proteinase K and Buffer T1 may be premixed directly before use Do not mix Buffer T1 Mix and Proteinase K more than 10 15 min before addition to the sample Proteinase K tends to self digestion in Buffer T1 without substrate 14 MACHEREY NAGEL 03 2014 Rev 13 NucleoSpin Tissue Incubate at 56 C until complete lysis is obtained at least 1 3 h Vortex occasionally during incubation or use a shaking incubator Samples can be incubated overnight as well If RNA free DNA is crucial for downstream applications a RNase digest may be performed Add 20 uL RNase A 10 mg mL solution not included see ordering information and incubate for an additional 5 min at room temperature Lyse sample Vortex the samples Add 200 uL Buffer B3 vortex vigorously and incubate at 70 C for 10 min Vortex briefly If insoluble particles are visible centrifuge for 5 min at high speed e g 11 000 x g and transfer the supernatant to anew microcentrifuge tube not provided Adjust DNA binding conditions Add 210 uL ethanol 96 100 to the sample and vortex vigorously After addition of ethanol a stringy precipitate may become visible This will not affect the DNA isolation Be sure to load all of the precipitate on the column in the following step Bind DNA For each sample place one NucleoSpin Tissue Column into a Collection Tube App
40. ume N acetyl cystein NaOH Vortex gently to mix Incubate the mixture for 25 min at room temperature with shaking Adjust the volume to 25 mL with sterile water Centrifuge for 30 min at 4 000 x g Discard the supernatant Resuspend the pellet in 0 5 1 mL Buffer T1 depending on sample viscosity Transfer 200 pL of the resuspended sample to a new microcentrifuge tube not provided Proceed with step 2 of the standard protocol see section 5 addition of Proteinase K and incubation MACHEREY NAGEL 03 2014 Rev 13 27 NucleoSpin Tissue 6 11 Support protocol for detection of EHEC bacteria in food e g fresh cows milk Before starting the preparation Check if Buffer B5 and Proteinase K were prepared according to section 3 Prepare mTSB and 3 2 M sodium acetate Set an incubator or water bath to 56 C Before elution preheat Elution Buffer BE to 70 C Prepare sample To a sterile 1 liter flask add 25 mL milk and 225 mL prewarmed 37 C mTSB medium supplied with Novobiocin Incubate the mixture in a shaking water bath for 5 6 h or overnight at 37 C Preparation of mTSB medium 30 g Tryptic Soy Broth Gibco 1 5 g bile salts No 3 Oxoid 1 5 g KHzPO Add 900 mL H O Filter the medium and adjust the pH with 2 M NaOH to 7 4 Add water to 1 liter and autoclave for 15 min at 121 C Centrifuge 100 mL culture for 40 min at 6 000 x g Gently pour off the supernatant and resuspend the pellet
41. xcept 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 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 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
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