Genomic DNA from Tissue
Contents
1. decreasing elution volume Figure 1 Correlation between elution volume and DNA concentration NucleoSpin Tissue XS Columns 8 MACHEREY NAGEL 10 2009 Rev 02 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 re sidual 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 yl The template may represent up to 4096 of the total PCR reaction volume if the eluate is incubated at elevated temperature as described above b A volume of 20 ul used for elution will evaporate to 12 14 ul during a heat incubation for 8 min at 90 C If a higher final volume is required please increase the volume of elution buffer e g from 20 ul to 30 pl c An incubation of the elution fraction for 8 min at 90 C will denature DNA I2. Genomic DNA from Tissue User Manual NucleoSpin Tissue XS October 2009 Rev 02 MACHEREY NAGEL MN Genomic DNA from Tissue Protocol at a glance Rev 02 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 4h 3 Lyse sample 80 ul B3 70 C 5 min 4 Adjust binding conditions 80 ul ethanol 5 Bind DNA Load lysate 1 min 11 000 x g 6 Wash silica 50 ul B5 membrane 1 wash 1 min 11 000 x g 50 yl B5 274 wash 2 min 11 000 x g 7 Elute DNA 20 ul BE 1 min gt 11 000 x g 8 Optional Optional Remove residual ethanol 90 C 8 min MACHEREY NAGEL GmbH amp Co KG Neumann Neander Str 6 8 D 52355 D ren Germany Tel 49 0 24 21 969 270 www mn net com e mail tech bio 9 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 risk and safety phrases 5 Protocols 5 1 Standard protocol for human or animal tissue 5 2 Protocol for cultured cells 5 8 Protocol for
3. for microcentrifuge tubes e Vortex mixer e Thermal heating block 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 my 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 MACHEREY NAGEL 10 2009 Rev 02 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 cre ated by adding ethanol The mixture is applied to the NucleoSpin Tissue XS Column and DNA binds to a silica membrane Two subsequent washing steps efficiently remove contaminati
4. lysate solution to a 1 5 ml microcentrifuge tube not provided Discard swab and continue with recovered solution 24 MACHEREY NAGEL 10 2009 Rev 02 NucleoSpin Tissue XS Lyse sample Add one volume of Buffer B3 200 400 ul vortex 2 x 5 s 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 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 10 2009 Rev 02 25 NucleoSpin Tissue XS 5 6 Support protocol for purification of genomic DNA from laser microdissected tissues 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 C 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 Prepare sample Place laser microdisseced sample into a 1 5 ml microcentrifuge tube not pro vided Pre lyse sample Add 80 pl Buf
5. paraffin embedded tissue 5 4 Support protocol for dried blood spots Guthrie cards 5 5 Support protocol for purification of genomic DNA from buccal swabs 5 6 Support protocol for purification of genomic DNA from laser microdissected tissues 5 7 Support protocol for blood samples 6 Appendix 6 1 Troubleshooting 6 2 Ordering information 6 3 Product use restriction warranty ana B A oN DD o 11 12 13 13 16 19 22 24 26 27 28 28 29 29 MACHEREY NAGEL 10 2009 Rev 02 Genomic DNA from Tissue 1 Components 1 1 Kit contents NucleoSpin Tissue XS 10 preps 50 preps 250 preps Cat No 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 B5 Concentrate aint am em Elution Buffer BE 5 ml 5 ml 15 ml Proteinase K lyophilized 6 mg 20 mg 2x50 mg Proteinase Buffer PB 0 8 ml 1 8 ml 8 ml NucleoSpin Tissue XS 10 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 HCl pH 8 5 4 MACHEREY NAGEL 10 2009 Rev 02 Genomic DNA from Tissue 1 2 Reagents consumables and equipment to be supplied by user Reagents e 96 100 ethanol Consumables e 1 5 ml microcentrifuge tubes for sample lysis and DNA elution e Disposable tips Equipment e Manual pipettors e Centrifuge
6. to remove droplets from tube lid MACHEREY NAGEL 10 2009 Rev 02 7 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 a reaction 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 slighty 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 elu tion volume and DNA concentration and will thus help you to find the optimized elution volume for your individual application NL MNS i I 15 i rd 2 103 2 3 i d i 2 i E 1 0 0 2 P d a 0 5 20 pl 10 pl 5 pl
7. 09 Rev 02 23 NucleoSpin Tissue XS 5 5 Support protocol for purification of genomic DNA from buccal swabs 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 C 25 C 1 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 2 Pre lyse sample Place the dry swab material in 1 5 ml microcentrifuge tubes not provided Add a mixture of 200 400 pl Buffer T1 and 20 40 yl Proteinase K solution 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 incuba tion 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 through Alternative B Transfer as much as possible of the
8. 15 min Adjust binding conditions Add 80 pl ethanol 96 100 to the lysate and mix by vortexing 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 Proceed with step 6 Wash silica membrane of the standard protocol see sec tion 5 1 MACHEREY NAGEL 10 2009 Rev 02 27 Genomic DNA from Tissue 6 Appendix 6 1 Troubleshooting Problem Possible cause and suggestions Low DNA content of the sample Low DNA yield e The content of DNA depends very much on sample type Column clogging amount and quality Sample contains residual cell debris or cells e 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 condi tions No increase of PCR signal despite of an increased volume of eluate used as template in PCR Residual ethanol in eluate Please see the detailed description of removal of residual traces of ethanol in section 2 6 Discrepancy between Aso quantification values and PCR quantification values Silica abrasion from the membrane e Due to the typically low DNA content in very small samples and the resulting low total amount of
9. 40902 50 740902 250 Wash Buffer B5 1 ml 2 ml 6 ml Concentrate Proteinase K Add 4 ml ethanol 6 mg Add 260 ul Proteinase Buffer Add 8 ml ethanol 20 mg Add 1 ml Proteinase Buffer Add 24 ml ethanol 2x50 mg Add 2 5 ml Proteinase Buffer to each vial MACHEREY NAGEL 10 2009 Rev 02 11 Genomic DNA from Tissue 4 Safety instructions risk and safety phrases 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 Component Hazard Hazard Risk Safety contents symbol phrases phrases B3 Guanidine x Xn Harmful if swal R 22 36 38 hydrochloride lowed Irritating to eyes and skin Proteinase K Proteinase K x Xn Irritating to eyes R 36 37 38 S 22 24 lyophilized Xi respiratory system 42 26 36 37 and skin may cause sensitization by inhalation Risk phrases R 22 Harmful if swallowed R 36 37 38 Irritating to eyes respiratory system and skin R 36 38 Irritating to eyes and skin R 42 May cause sensitization by inhalation Safety phrases S22 Do not breathe dust S24 Avoid contact with the skin S 26 In case of contact with eyes rinse immediately with plenty of water and seek medical advice S 36 37 Wear suitable protective clothing and gloves Hazard labeling not neccessary if quantity per bottle below 125 g or ml certificate of exemption accor
10. 5 min 16 MACHEREY NAGEL 10 2009 Rev 02 NucleoSpin Tissue XS Adjust binding conditions Add 80 pl ethanol 96 100 to the lysate and mix by 80 pl vortexing 2 x 5 s ethanol Spin down briefly to clear the lid Bind DNA For each sample place one NucleoSpin Tissue XS RE Column into a Collection Tube 2 ml Apply the sample Load lysate to the column Centrifuge for 1min at 11 000 x g Discard the flow through and place the column into a new Collection Tube 2 ml es 1 min 11 000x g If the sample is not drawn completely through the matrix re peat the centrifugation step at 11 000 x g Wash silica membrane 50 ul B5 EI u T Add 50 pl Buffer B5 to NucleoSpin Tissue XS Column 11 000 x g Centrifuge for 1 min at 11 000 x g It is not necessary to discard the flow through Reuse the Collection Tube EXT ais Add 50 pl Buffer B5 directly onto the membrane of the 2 min NucleoSpin Tissue XS Column Centrifuge for 2 min at 11 000 x g 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 pl 20 ul BE 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 pl Tin For a correlation of elution volume DNA concentration and 11 000 x g DNA amount eluted from the column see se
11. com for more detailed product information 6 3 Product use restriction warranty NucleoSpin Tissue XS kit components were developed designed distributed and sold FOR RESEARCH PURPOSES ONLY They are suitable FOR IN VITRO USES ONLY No claim or representation is intended for its use to identify any specific organ ism or for clinical use diagnostic prognostic therapeutic or blood banking It is rather the responsibility of the user to verify the use of the NucleoSpin Tissue XS kit for a specific application range as the performance characteristic of this kit has not been verified to a specific organism This MACHEREY NAGEL product is shipped with documentation stating specifica tions and other technical information MACHEREY NAGEL warrants to meet the stated specifications MACHEREY NAGEL Ss sole obligation and the customer s sole remedy is limited to replacement of products free of charge in the event products fail to perform as warranted Supplementary reference is made to the general business terms and conditions of MACHEREY NAGEL which are printed on the price list Please contact us if you wish an extra copy MACHEREY NAGEL 10 2009 Rev 02 29 Genomic DNA from Tissue MACHEREY NAGEL does not warrant against damages or defects arising in shipping and handling transport insurance for customers excluded or out of accident or im proper or abnormal use of this product against defects in products or components not manufa
12. ction 2 4 2 5 MACHEREY NAGEL 10 2009 Rev 02 17 NucleoSpin Tissue XS Optional Remove residual ethanol gt Optional Incubate elution fraction with open lid for 8 min at 90 C min See section 2 5 for further comments and alternative in 5 0 C cubation times and temperatures for a removal of residual ethanol 18 MACHEREY NAGEL 10 2009 Rev 02 NucleoSpin Tissue XS 5 3 Protocol for paraffin embedded tissue Prepare sample 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 twee zers or toothpicks and place the samples into microcen trifuge tubes not provided Add 300 pl 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 superna tant 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 4 should be doubled 160 pl each However also for larger sampl
13. ctured by MACHEREY NAGEL or against damages resulting from such non MACHEREY NAGEL components or products MACHEREY NAGEL makes no other warranty of any kind whatsoever and SPECIFICALLY DISCLAIMS AND EXCLUDES ALL OTHER WARRANTIES OF ANY KIND OR NATURE WHATSOEVER DIRECTLY OR INDIRECTLY EXPRESS OR IMPLIED INCLUDING WITHOUT LIMITATION AS TO THE SUITABILITY REPRODUCTIVITY DURABILITY FITNESS FOR A PARTICULAR PURPOSE OR USE MERCHANTABILITY CONDITION OR ANY OTHER MATTER WITH RESPECT TO MACHEREY NAGEL PRODUCTS In no event shall MACHEREY NAGEL be liable for claims for any other damages whether direct indirect incidental compensatory foreseeable consequential or spe cial including but not limited to loss of use revenue or profit whether based upon warranty contract tort including negligence or strict liability arising in connection with the sale or the failure of MACHEREY NAGEL products to perform in accordance with the stated specifications This warranty is exclusive and MACHEREY NAGEL makes no other warranty expressed or implied The warranty provided herein and the data specifications and descriptions of this MACHEREY NAGEL product appearing in MACHEREY NAGEL published catalogues and product literature are MACHEREY NAGEL s sole representations concerning the product and warranty No other statements or representations written or oral by MACHEREY NAGEL s employees agent or representatives except written state ments
14. ding to 67 548 EEC Art 25 1999 45 EC Art 12 and German GefStoffV 8 20 3 and TRGS 200 7 1 For further information see Material Safety Data Sheet 12 MACHEREY NAGEL 10 2009 Rev 02 NucleoSpin Tissue XS 5 Protocols 5 1 Standard protocol for human or animal tissue Before starting the preparation e Check if Wash Buffer B5 and Proteinase K were prepared according to section 3 e Adjust thermal heating block temperature to 56 C and equilibrate sample to room temperature 18 C 25 C 1 Prepare sample Place the sample of up to 2 5 mg into a 1 5 ml microcen trifuge tube not provided For samples from 2 5 10 mg double the volumes of Buffer T1 Buffer B3 and ethanol in steps 2 3 4 to 160 ul each 2 Pre lyse sample Add 80 ul Buffer T1 and 8 pl Proteinase K solution and mix by vortexing 2 x 5 s Be sure that the sample is com 80 ul T1 pletely covered with lysis solution 8 pl 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 approxi 1 4h mately 1 4 h or overnight Vortex occasionally during incubation or use a shaking incubator At the end of the or incubation adjust the thermal heating block temperature to 70 C f
15. e e g laser microdissected fresh frozen Blood samples 1 30 ul fresh or frozen blood Typical sample size 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 Typical yields for selected samples are listed below 100 HeLa cells 0 1 0 5 ng DNA 1000 HeLa cells 1 5 ng DNA Typical yield 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 Elution volume 5 30 ul Approximatively 40 45 min for 6 12 samples Preparation tim ke Spara exclusive incubation for lysis Format XS spin columns 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 per forming sampling and DNA purification with special care in order to avoid a contamina tion 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 e Wear personal protection equipment lab coat gloves goggles e Use aerosol resistant pipette tips e Always change pipette tips between liquid transfers Briefly centrifuge after mixing steps in order
16. 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 10 2009 Rev 02 31
17. entrifugation step at 11 000 x g 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 through Reuse the Collection Tube y 80 pl B3 70 C 5 min 80 pl ethanol Load lysate 1 min 11 000 x g 50 pl B5 1 min 11 000 x g 14 MACHEREY NAGEL 10 2009 Rev 02 NucleoSpin Tissue XS Add 50 pl 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 pl 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 pl 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 ul B5 2 min 11 000 x g 20 pl BE 1 min 11 000x g Optional 8 min 90 C MACHEREY NAGEL 10 2009 Rev 02 15 NucleoSpin Tissue XS 5 2 Protocol for cultured cells Before starting the preparation Check if Wash Buffer B5 a
18. es the indicated volume 300 ul of n octane or xylene can be used MACHEREY NAGEL 10 2009 Rev 02 19 NucleoSpin Tissue XS Pre lyse sample Add 80 ul Buffer T1 and 8 pl Proteinase K solution and mix by vortexing 2 x 5 s Be sure that the sample is com pletely 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 lysis is obtained approxi matively 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 2 x 5 s 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 cen trifuge for 5 min at high speed e g 11 000 x g and transfer the supernatant to a new microcentrif
19. f non denatured DNA is required for downstream applications other than PCR e g 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 d 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 e Ifthe 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 e g 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 10 2009 Rev 02 9 Genomic DNA from Tissue 25 without shaking dk 700 rpm 1400 rpm bs eo a Incubation time min o 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 inc
20. fer T1 and 8 pl Proteinase K solution Mix by vortexing 2 5 s and incubate 10 min at 56 C for approximately 1 4 h or overnight The optimal incubation time may vary depending on sample type and amount Lyse sample Add 80 yl Buffer B3 vortex 2 x 5 s and incubate at 70 C for 5 min Vortex briefly after the incubation Let the lysate cool down to room temperature Adjust binding conditions Add 80 yl ethanol 96 100926 to each 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 26 MACHEREY NAGEL 10 2009 Rev 02 NucleoSpin Tissue XS 5 7 Support protocol for blood samples 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 C 25 C Prepare sample Not necessary Lyse blood samples Pipette 8 pl Proteinase K solution and up to 20 pl blood into a 1 5 ml microcen trifuge tube not provided Add 60 pl 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 Lyse sample Add 80 pl Buffer B3 to the sample and vortex the mixture vigorously 10 20 s Incubate samples at 70 C for 10
21. isolated DNA a DNA quantification via A absorption measuerment is often hampered due to the low sensitivity of the absorp tion measuement When performing absorption measure ments close to the detection limit of the photometer the measurement may be influenced by minor amounts of silica abrasion In order to prevent incorrect A quanti fication of small DNA amounts centrifuge the eluate for 30 s at gt 11 000 x g and take an aliquot for measuement without disturbing any sediment Alternatively use a sili ca abrasion insensitive DNA quantification method e g PicoGreen fluorecent dye Unexpected A ud Aso ratio Measurement not in the range of photometer detection limit e In order to obtain a significant A A ratio it is necessary that the initially measured A and A values are signifi cantly above the detection limit of the photometer used An A value close to the background noise of the pho tometer will cause unexpeced A A ratios 28 MACHEREY NAGEL 10 2009 Rev 02 Genomic DNA from Tissue 6 2 Ordering information Product Cat No Pack of NucleoSpin Tissue XS 740901 10 50 250 10 50 250 Buffer T1 740940 25 25 ml Buffer B3 740920 100 ml Buffer B5 Concentrate For 100 ml Buffer B5 740921 20ml Buffer BE 740306 100 100 ml Proteinase K 740506 100 mg NucleoSave 740403 10 100 10 100 NucleoSpin Filters 740606 50 Collection Tubes 2 ml 740600 1000 Visit www mn net
22. king incubator Adjust the thermal 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 22 MACHEREY NAGEL 10 2009 Rev 02 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 a 1 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 Lysesample Add 160 pl 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 160 yl 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 10 20
23. me may be varied from approximately 5 30 pl 1 fe Ld For a correlation of elution volume DNA concentration and 1000 x g DNA amount eluted from the column see section 2 4 2 5 8 Optional Remove residual ethanol Optional Incubate elution fraction with open lid for 8 min at 90 C min See section 2 5 for further comments and alternative in pis cubation times and temperatures for a removal of residual ethanol MACHEREY NAGEL 10 2009 Rev 02 21 NucleoSpin Tissue XS 5 4 Support 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 C 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 pl 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 sha
24. nd Proteinase K were prepared according to section 3 Adjust thermal heating block temperature to 56 C and equilibrate sample to room temperature 18 C 25 C 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 2xbs 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 2 x 5 s 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 V a 80 pl T1 8 pl Proteinase K 56 C 10 min 80 ul B3 70 C
25. ons 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 e 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 NucleoSave FTA cards see ordering on formation buccal swabs forensic samples and others see specifications at a glance Table 1 page 7 e 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 e The DNA yield strongly depends on the sample type quality and amount see specifications at a glance Table 1 page 7 e 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 10 2009 Rev 02 Genomic DNA from Tissue Table 1 Kit specifications at a glance Parameter NucleoSpin Tissue XS Tissue samples 0 025 10 mg tissu
26. or 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 ordering information and incubate for additional 5 min at room temperature MACHEREY NAGEL 10 2009 Rev 02 18 NucleoSpin Tissue XS Lyse sample Add 80 ul Buffer B3 vortex 2 x 5 s 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 pl ethanol 96 100 to the lysate and mix by vortexing 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 1min 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 c
27. signed by a duly authorized officer of MACHEREY NAGEL are authorized they should not be relied upon by the customer and are not a part of the contract of sale or of this warranty Product claims are subject to change Therefore please contact our Technical Service Team for the most up to date information on MACHEREY NAGEL products You may also contact your local distributor for general scientific information Applications mentioned in MACHEREY NAGEL literature are provided for informational purposes only MACHEREY NAGEL does not warrant that all applications have been tested in MACHEREY NAGEL laboratories using MACHEREY NAGEL products MACHEREY NAGEL does not warrant the correctness of any of those applications Please contact MACHEREY NAGEL Germany Tel 49 0 24 21 969 270 e mail TECH BIO mn net com Last updated 12 2006 Rev 02 30 MACHEREY NAGEL 10 2009 Rev 02 Genomic DNA from Tissue Trademarks Dacron is a trademark of Daigger DyNAmo is a trademark of Finnzymes Oy LightCycler is a trademark of a member of the Roche Group NucleoSpin is a trademark of MACHEREY NAGEL GmbH amp Co KG PicoGreen is a registered trademark of Molecular Probes Inc SYBR is a registered trademark 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
28. ubation of the eluate is recom mended Heat incubation may be performed at temperatures of 70 90 C in a 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 10 2009 Rev 02 Genomic DNA from Tissue 3 Storage conditions and preparation of working solutions Attention Buffers B3 and B5 contain guanidine hydrochloride Wear gloves and goggles e All kit components can be stored at room temperature 20 25 C and are stable up to one year e 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 96 10096 to Buffer C5 Concentrate Mark the label of the bottle to indicate that ethanol was added Store Wash Buffer C5 at room temperature 20 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 Cat No 740902 10 7
29. uge tube not provid ed Adjust binding conditions Add 80 pl ethanol 96 100 to the lysate and mix by vortexing 2 x 5 s Spin down briefly to clear the lid ei 80 ul T1 8 yl Proteinase K 56 C 1 4h or 56 C overnight 80 ul B3 70 C 5 min 80 pl ethanol 20 MACHEREY NAGEL 10 2009 Rev 02 NucleoSpin Tissue XS 5 Bind DNA For each sample place one NucleoSpin Tissue XS El Column into a Collection Tube 2 ml Apply the sample z Load lysate to the column Centrifuge for 1min at 11 000 x g Discard the flow through and place the column into a new Collection Tube 2 ml e 1 min 11 000 x g If the sample is not drawn completely through the matrix re peat the centrifugation step at 11 000 x g 6 Wash silica membrane 50 ul B5 ETT iii Add 50 pl Buffer B5 to NucleoSpin Tissue XS Column 11 000 x g Centrifuge for 1 min at 11 000 x g It is not necessary to discard the flow through Reuse the Collection Tube 2 wash ga Sap Add 50 pl Buffer B5 directly onto the membrane of the 2 min NucleoSpin Tissue XS Column Centrifuge for 2 min at 11 000 x g 11 000 x g Discard Collection Tube with flow through 7 Elute DNA Place the NucleoSpin Tissue XS Column in a new 1 5 ml microcentrifuge tube not provided and apply 20 pl 20 ul BE Buffer BE directly onto the center of the silica membrane of the column Centrifuge for 1 min at 11 000 x g Elution volu
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