Genomic DNA from Tissue
Contents
1. Please contact MACHEREY NAGEL Germany Tel 49 0 24 21 969 270 e mail TECH BIO Q mn net com Last updated 12 2006 Rev 02 Trademarks Dacron is a trademark of E I du Pont de Nemours and Company FTA is a trademark of Whatman Ltd NucleoSpin is a trademark of MACHEREY NAGEL GmbH amp Co KG All used names and denotations can be brands trademarks or registered labels of their respective owner also if they are not special denotation To mention products and brands is only a kind of information i e it does not offend against trademarks and brands and can not be seen as a kind of recommendation or assessment Regarding these products or services we can not grant any guarantees regarding selection efficiency or operation 38 MACHEREY NAGEL 01 2010 Rev 112. Genomic DNA from Tissue User Manual NucleoSpin Tissue January 2010 Rev 11 MACHEREY NAGEL MN Genomic DNA Purification from Tissue Protocol at a glance Rev 11 NucleoSpin Tissue 1 Prepare sample Cut 25 mg into small pieces Pre lyse sample 180 ul T1 25 ul Proteinase K 56 C 1 3h Lyse sample 200 yl B3 70 C 10 min Adjust DNA binding conditions 210 ul 96 100 ethanol Bind DNA Wash silica membrane Dry silica membrane Load all 11 000 x g 1 min 1 wash 500 ul BW 24 wash 600 ul B5 11 000 x g 1 min 11 000x g 1 min Elute highly pure DNA cx 11 000 x g 1 min 100 pl BE 70 C RT 1 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 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 f 2 2 Kit specifications 7 2 3 Elution procedures 8 3 Storage conditions and preparation of working solutions 9 4 Safety instructions risk and safety phrases 10 5 Standard protocol for human or animal tissue and cultured cells 11 6 Support protocols 14 6 1 Support protocol for mouse or rat tails 14 6 2 Support protocol for bacteria 15 6 3 Suppor
3. 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 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 Lyse sample Vortex the samples Add 200 yl Buffer B3 vortex vigor ously 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 a new microcentrifuge tube not provided Adjust DNA binding conditions Add 210 pl 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 Apply 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 x g Discard flow through GB c 56 C 1 3h or 56 C overnight 200 pl B3
4. 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 shak ing 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 infor mation 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 lyso staphin 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 1 MACHEREY NAGEL 01 2010 Rev 11 15 NucleoSpin Tissue 6 3 Support protocol for yeast Before starting the preparation Check if Buffer B3 Buffer B5 and Proteinase K were prepared according to section 3 Check that sorbitol buffer and lyticase or zymolase not provide
5. 70 C 10 min 210 ul ethanol Load samples 11 000 x g 1 min 12 MACHEREY NAGEL 01 2010 Rev 11 NucleoSpin Tissue 6 Wash silica membrane Add 500 ul Buffer BW Centrifuge for1 minat 11 000 x g Discard flow through and place the column back into the Collection Tube Add 600 pl Buffer B5 to the column and centrifuge for 1 min at 11 000 x g Discard flow through and place the column back into the Collection Tube 6 Dry silica membrane Centrifuge the column for 1 min at 11 000 x g Residual ethanol is removed during this step 7 Elute highly pure DNA Place the NucleoSpin Tissue Column into a 1 5 ml microcentrifuge tube not provided and add 100 pl pre warmed Buffer BE 70 C Incubate at room tempera ture for 1 min Centrifuge 1 min at 11 000 x g For alternative elution procedures see section 2 3 500 ul BW 11 000x g F 1 min e 600 pI BS 11 000 x g 1 min 11 000 x g 1 min 100 ul BE 70 C RT 1 min ed 11 000 x g 1 min MACHEREY NAGEL 01 2010 Rev 11 18 NucleoSpin Tissue 6 6 1 Support protocols Support protocol for mouse or rat tails Before starting the preparation Check if Buffer B3 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 pl
6. 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 Bind DNA Transfer 600 pl of the samples from the 2 ml microcentrifuge tubes into NucleoSpin Tissue Columns Centrifuge at 11 000 x g for 1 min 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 1 MACHEREY NAGEL 01 2010 Rev 11 33 Genomic DNA from Tissue 7 Appendix 7 1 Troubleshooting Problem Possible cause and suggestions Incomplete lysis e 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 e Decreased Proteinase K activity Store dissolved Proteinase K at 20 C for 6 months Reagents not applied properly e Prepare Buffer B3 BufferB5 and Proteinase K solution according to instructions see section 3 Add ethanol to the No or poor lysates before loading them onto the columns DNA yield Suboptimal elution of DNA from the column e Preheat Buffer BE to 70 C before elution Apply Buffer BE directly onto the center of the silica membrane e Elution efficiencies decrease dramatic
7. K Two possible working procedures A 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 resus pended solution of the second tube to the third tube and so on Finally continue with step 3 B Resuspend each pellet as mentioned above and proceed with step 3 In this case the solutions are pooled and the spin column has to be loaded succes sively 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 pl ethanol 96 100 to the sample and vortex vigorously 28 MACHEREY NAGEL 01 2010 Rev 11 NucleoSpin Tissue 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 Wash silica membrane Add 500 yl 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 pl Buffer B5 to the column and centrifuge for 2 min at 11 000 x g Discard flow through and place 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
8. NucleoSpin Tissue 5 Standard protocol for human or animal tissue and cultured cells Before starting the preparation e Check if Buffer B3 Buffer B5 and Proteinase K were prepared according to section 3 e Set an incubator or water bath to 56 C e Preheat Elution Buffer BE to 70 C 1 Prepare sample Tissue 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 Polytron Ultra Turrax Add 25 mg of tissue to a 1 5 ml microcentrifuge tube not provided add 50 75 ul phos phate 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 ul Buffer B3 Incubate the sample at 70 C for 10 15 min Proceed with step 4 2 Pre lyse sample Add 180 pl Buffer T1 and 25 pl Proteinase K solution Vortex to mix Be sure that the samples are completely 180 ul T1 covered with lysis solution y 25 ul If processing several samples Proteinase K and Buffer T1 Proteinase K may be premixed directly before use Do never mix Buffer T1 and Proteinase K more than 10 15 min before addition to the sample Proteinase K tends to self digestion in Buffer T1 without substrate MACHEREY NAGEL 01 2010 Rev 11 11 NucleoSpin
9. and wash the pellet with 1 ml 70 ethanol and resuspend in 10 pl sterile water MACHEREY NAGEL 01 2010 Rev 11 25 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 e Check if Buffer B3 Buffer B5 and Proteinase K were prepared according to section 3 e Set an incubator or water bath to 56 C Before elution preheat Elution Buffer BE to 70 C 1 Prepare sample e 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 Proceed with step 2 of the standard protocol see section 5 1 26 MACHEREY NAGEL 01 2010 Rev 11 NucleoSpin Tissue 6 13 Support protocol for purification of bacterial DNA e g Borrelia burgdorferi from urine Before starting the preparation Check if Buffer B3 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 m
10. con tents Wear gloves and goggles and follow the safety instructions given in this section Component Hazard Hazard Risk Safety contents symbol phrases phrases B1 Guanidine x Xn Harmful if swal R 22 hydrochloride lowed Irritating to 36 38 eyes and skin BW Guanidine x Xn Flammable Harmful R 10 22 S 7 16 25 hydrochloride if swallowed 36 38 isopropanol Irritating to eyes and lt 25 skin Proteinase K Proteinase K x Xn Irritating to eyes R S 22 24 lyophilized Xi respiratory system 36 37 38 26 36 37 and skin may cause 42 sensitization by inhalation Risk phrases R 10 Flammable R22 Harmful if swallowed R 36 37 88 Irritating to eyes respiratory system and skin R 36 38 Irritating to eyes and skin R42 May cause sensitization by inhalation Safety phrases S7 Keep container tightly closed S 16 Keep away from sources of ignition No smoking S22 Do not breathe dust S24 Avoid contact with the skin S25 Avoid contact with the eyes 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 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 10 MACHEREY NAGEL 01 2010 Rev 11
11. 0 preps 250 preps Cat No 740952 10 740952 50 740952 250 Lysis Buffer T1 5 ml 20 ml 100 ml Buffer B1 6 ml 12 ml 60 ml Buffer B2 1 5 ml 3ml 15 ml Wash Buffer BW 6 ml 30 ml 2x75ml deles 4 mi 2x7ml 2 x 40 ml Elution Buffer BE 3 ml 15 ml 75 ml pamai 6 mg 30 mg 2x75mg Proteinase Buffer PB 0 8 ml 1 8 ml 8ml em E TRUE rings 18 39 239 Collection Tubes 2 ml 20 100 500 Labels for Lysis Buffer B3 1 1 1 User Manual 1 1 1 For preparation of working solutions and storage conditions see section 3 Composition of Elution Buffer BE 5 mM Tris HCI pH 8 5 MACHEREY NAGEL 01 2010 Rev 11 5 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 for microcentrifuge tubes e Vortex mixer e Heating block for incubation at 70 C e Equipment for sample disruption and homogenization see section 2 5 e 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 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 purifi
12. 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 Depending on the bacterial strains that are to be detected incubation at 95 C can be skipped 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 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 1 MACHEREY NAGEL 01 2010 Rev 11 31 NucleoSpin Tissue 6 17 Support protocol for purification of genomic DNA from buccal swabs Before starting the preparation Check if Buffer B3 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 Collect the samples with cotton dacron Daigger or C E P swabs Gibco BRL Scra
13. a Transfer 200 pl 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 1 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 typi cally change due to the increased release of bacterial pathogen DNA MACHEREY NAGEL 01 2010 Rev 11 21 NucleoSpin Tissue 6 9 Support protocol for viral DNA e g CMV from stool Before starting the preparation Check if Buffer B3 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 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 4x1 mlina 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 filt
14. ace 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 pl Buffer T1 and 25 yl Proteinase K and vortex Incubate at 56 C overnight or until complete lysis is obtained Vortex occasionally during incuba tion 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 sub strate 3 Lyse sample Add 200 pl 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 pl ethanol to the lysate and vortex vigorously Proceed with step 5 of the standard protocol see section 5 1 14 MACHEREY NAGEL 01 2010 Rev 11 NucleoSpin Tissue 6 2 Support protocol for bacteria Before starting the preparation Check if Buffer B3 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
15. ally if elution is done with buffers with a pH lt 7 0 Use slightly alkaline elution buffers like Buffer BE pH 8 5 e 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 e 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 34 MACHEREY NAGEL 01 2010 Rev 11 Genomic DNA from Tissue Reagents not applied properly e Prepare Buffer B3 Buffer B5 and Proteinase K solution accord ing to instructions see section 3 Add ethanol to the lysates Poor DNA before loading them on the columns quality continued RNA in sample e f RNA free DNA is desired add 10 ul of RNase A solution 5 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 e Do not use more sample material than recommended 25 mg for most tissue types If insoluble material like bones or hair remains in the lysate spin down the debris and transfer the clear supernatant to a new microcentrifuge tube before pro ceeding with addition of Buffer B3 and ethanol Incomplete lysis Clogged e Sample not thorou
16. and incubate for further 3 5 min at 70 C Centrifuge for 1 min at 4 500 x g MACHEREY NAGEL 01 2010 Rev 11 29 NucleoSpin Tissue 6 15 Support protocol for purification of genomic DNA from insects Before starting the preparation e Check if Buffer B3 Buffer B5 and Proteinase K were prepared according to section 3 e 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 1 30 MACHEREY NAGEL 01 2010 Rev 11 NucleoSpin Tissue 6 16 Support protocol for purification of genomic DNA from dental swabs Before starting the preparation Check if Buffer B3 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 Place swab material paper cotton brushes plastic in a 1 5 ml microcentrifuge tube not provided Pre lyse sample Add 180 pl Buffer T1 and 25 ul Proteinase 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
17. 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 1 20 MACHEREY NAGEL 01 2010 Rev 11 NucleoSpin Tissue 6 8 Support protocol for genomic DNA from stool Before starting the preparation e Check if Buffer B3 Buffer B5 and Proteinase K were prepared according to section 3 e Set an incubator or water bath to 56 C e Before elution preheat Elution Buffer BE to 70 C 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 bacteri
18. ca tion procedure All technical literature is available on the internet at www mn net com 6 MACHEREY NAGEL 01 2010 Rev 11 Genomic DNA from Tissue 2 2 1 Product description 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 binding of DNA to the silica membrane in the NucleoSpin Tissue Columns are created 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 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 A A 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 bacte
19. d Collection Tube with flow through Proceed with step 6 Wash silica membrane of the standard protocol see section 5 1 18 MACHEREY NAGEL 01 2010 Rev 11 NucleoSpin Tissue 6 6 Support protocol for hair roots Before starting the preparation Check if Buffer B3 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 off the hair roots from the hair sample up to 100 and collect them in a 1 5 ml microcentrifuge tube not provided 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 1 MACHEREY NAGEL 01 2010 Rev 11 19 NucleoSpin Tissue 6 7 Support protocol for paraffin embedded tissue Before starting the preparation Check if Buffer B3 Buffer B5 and Proteinase K were prepared according to section 3 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
20. d 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 1 Prepare sample Harvest 3 ml YPD yeast culture OD 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 su pernatant and pellet the cells by centrifugation 5 000 x g 10 min 2 Pre lyse sample Resuspend the pellet in 600 pl sorbitol buffer 1 2 M sorbitol 10 mM CaCl 0 1M Tris HCl pH 7 5 35 mM f mercaptoethanol Add 50 U lyticase 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 g remove supernatant and resus pend the pelleted spheroplasts in 180 pl Buffer T1 Add 25 pl Proteinase K solution and vortex vigorously Incubate at 56 C until complete lysis is obtained at least 1 3 h Vortex occasionally during incubation 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 infor mation and incubate for an additional 5 min at room temperature Proceed with step 3 of the standard protocol see section 5 1 Other protocols use 5 200 U lyticase or zymolase depending on enzyme q
21. ded Proceed with step 2 of the standard protocol see section 5 1 24 MACHEREY NAGEL 01 2010 Rev 11 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 B3 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 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 KH PO 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 in 2 ml sterile water Centrifuge for 10 min at 10 000 x g Pre lyse sample Resuspend the pellet in 180 pl Buffer T1 and add 25 pl Proteinase K solution Carry out the standard protocol beginning with step 3 see section 5 1 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 pl eluate Centrifuge for 30 min at 11 000 x g Discard supernatant
22. e up to one year During storage especially at low temperatures a white precipitate may form in Buffer T1 B1 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 Lysis Buffer B3 Transfer the total contents of Buffer B1 to Buffer B2 and mix well Place the labels for Lysis Buffer B3 on the bottle The resulting Lysis Buffer B3 is stable for up to one year at room temperature 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 Store Wash Buffer B5 at room temperature 18 25 C for up to one year Proteinase K Add the indicated volume of Proteinase Buffer PB to dissolve lyophilized Proteinase K Proteinase K solution is stable at 20 C for up to 6 months NucleoSpin Tissue 10 preps 50 preps 250 preps Cat No 740952 10 740952 50 740952 250 Wash 4 ml 2x7ml 2 x40 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 260 ul Add 1 35 ml Add 3 35 ml Proteinase Buffer Proteinase Buffer Proteinase Buffer to each vial MACHEREY NAGEL 01 2010 Rev 11 9 NucleoSpin Tissue 4 Safety instructions risk and safety phrases The following components of the NucleoSpin Tissue kits contain hazardous
23. efore starting the preparation Check if Buffer B3 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 Lyse blood sample Pipette 25 ul Proteinase K and up to 200 pl 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 l 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 vigor ously during incubation if processing older or clotted blood samples Adjust DNA binding conditions Add 210 pl ethanol 96 100 to each sample and vortex again 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 15 000 x g Discar
24. ghly homogenized and mixed with Buffer T1 columns Proteinase K The mixture has to be vortexed vigorously imme diately after the addition of Buffer T1 e Decreased Proteinase K activity Store dissolved Proteinase K at 20 C for 6 months Reagents not applied properly e Prepare Buffer B3 Buffer B5 and Proteinase K solution accord ing to instructions see section 3 Add ethanol to the lysates before loading them on the columns Carry over of ethanol or salt Suboptimal e Make sure to centrifuge 1 min at 11 000 x gin order to remove performance all of ethanolic Buffer B5 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 e Do not chill Buffer B5 before use Cold buffer will not remove reactions salt effectively Equilibrate Buffer B5 to room temperature before use MACHEREY NAGEL 01 2010 Rev 11 35 Genomic DNA from Tissue Suboptimal performance of genomic DNA in enzymatic reactions continued Contamination of DNA with inhibitory substances Do not elute DNA with TE buffer EDTA may inhibit enzymatic reactions Repurify DNA and elute in Buffer BE If the A A ratio of the eluate is below 1 6 repeat the purifica tion 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 standard prot
25. l 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 Repeat this steps a third time The sample material should be fresh and storage at 20 C to 80 C is only recom mended 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 1 MACHEREY NAGEL 01 2010 Rev 11 27 NucleoSpin Tissue 6 14 Support protocol for purification of viral DNA e g CMV from urine Before starting the preparation Check if Buffer B3 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 4x 1 ml in a 1 5 ml microcentrifuge tube Carefully decant 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 A0 C If a complete solution does not happen let the precipitate sediment and proceed with step 1 of the support protocol using only the superna tant 2 Pre lyse sample Resuspend the pellet in 180 pl Buffer T1 and 25 pl Proteinase
26. ntract 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 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 MACHEREY NAGEL 01 2010 Rev 11 37 Genomic DNA from Tissue 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
27. ocol see section 5 1 7 2 Ordering information Product Cat No Pack of NucleoSpin Tissue NucleoSpin Tissue XS NucleoSpin Blood Buffer T1 Buffer B3 Buffer B5 Concentrate for 100 ml Buffer B5 Buffer BW Proteinase K HNase A NucleoSpin DNA Trace Collection Tubes 2 ml NucleoCard Visit www mn net com for more detailed product information 740952 10 50 250 740901 10 50 250 740951 10 50 250 740940 25 740920 740921 740922 740506 740505 50 740505 740942 4 25 740600 740403 10 100 10 50 250 preps 10 50 250 preps 10 50 250 preps 25 ml 100 ml 20 ml 100 ml 100 mg 50 mg 4 25 preps 1000 10 100 cards 36 MACHEREY NAGEL 01 2010 Rev 11 Genomic DNA from Tissue 7 3 Product use restriction warranty NucleoSpin Tissue 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 organism 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 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
28. pe 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 2 Pre lyse sample Place the dry swab material in 2 ml microcentrifuge tubes not provided Add 400 600 pl 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 pl are necessary Mix by vortexing 2 x 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 lysate solution to a 1 5 ml microcentrifuge tube not provided Discard swab and continue with recovered solution 32 MACHEREY NAGEL 01 2010 Rev 11 NucleoSpin Tissue Lyse sample Add one volume Buffer B3 400 or 600 ul 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 Adjust
29. rate and centrifuge for 1 min at 11 000 x g Pre lyse sample Carefully remove the supernatant by decanting Add 400 pl Buffer T1 and 35 pl 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 22 MACHEREY NAGEL 01 2010 Rev 11 NucleoSpin Tissue 6 Wash silica membrane Add 600 yl 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 pl Buffer B5 to the column and centrifuge for 1 min at 4 500 x g Discard flow through and place the column back into the Collection Tube Add 600 pl Buffer B5 to the column and centrifuge for 2 min at 11 000 x g Discard flow through 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 Elute highly pure DNA Place the N
30. rial 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 Sample material 1 25 mg 10 10 cells Typical yield 20 35 ug Elution volume 60 100 ul Binding capacity 60 ug Preparation time 20 min 4 6 preps after lysis Format Mini spin column MACHEREY NAGEL 01 2010 Rev 11 7 Genomic DNA from Tissue 2 3 Elution procedures In addition to the standard method recovery rate about 70 90 several modifica tions 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 10096 of bound nucleic acid can be eluted High concentration Perform one elution step with 6096 of the volume indicat ed in the individual protocol Concentration of DNA will be ca 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 individual protocol incubate for 3 min and centrifuge Apply a second aliquot of elution buffer incubate and centrifuge again Thus about 85 100 of bound nucleic acid is eluted in the standard elution volume at a high concen
31. t protocol for yeast 16 6 4 Support protocol for dried blood spots e g NucleoCards FTA cards Guthrie cards 17 6 5 Support protocol for genomic DNA and viral DNA from blood samples 18 6 6 Support protocol for hair roots 19 6 7 Support protocol for paraffin embedded tissue 20 6 8 Support protocol for genomic DNA from stool 21 6 9 Support protocol for viral DNA e g CMV from stool 22 6 10 Support protocol for detection of Mycobacterium tuberculosis or Legionella pneumophila in sputum or bronchoalveolar lavage 24 6 11 Support protocol for detection of EHEC bacteria in food e g fresh cows milk 25 6 12 Support protocol for purification of bacterial DNA e g Chlamydia trachomatis from cultures biological fluids or clinical specimens 26 6 13 Support protocol for purification of bacterial DNA e g Borrelia burgdorferi from urine 27 6 14 Support protocol for purification of viral DNA e g CMV from urine 28 MACHEREY NAGEL 01 2010 Rev 11 3 Genomic DNA from Tissue 6 15 Support protocol for purification of genomic DNA from insects 30 6 16 Support protocol for purification of genomic DNA from dental swabs 31 6 17 Support protocol for purification of genomic DNA from buccal swabs 32 7 Appendix 34 7 1 Troubleshooting 34 7 2 Ordering information 36 7 3 Product use restriction warranty 37 4 MACHEREY NAGEL 01 2010 Rev 11 Genomic DNA from Tissue 1 Components 1 1 Kit contents NucleoSpin Tissue 10 preps 5
32. 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 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 manufactured 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 co
33. tration Convenient elution For convenience elution buffer of ambient temperature may be used This will result in a somewhat lower yield approximately 2096 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 elution 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 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 8 MACHEREY NAGEL 01 2010 Rev 11 Genomic DNA from Tissue 3 Storage conditions and preparation of working solutions Attention Buffers B1 B3 and BW contain guanidine hydrochloride Wear gloves and goggles All kit components can be stored at room temperature 18 25 C and are stabl
34. uality or brand Increasing the enzyme concentration may be required if spheroplasts are not formed 16 MACHEREY NAGEL 01 2010 Rev 11 NucleoSpin Tissue 6 4 Support protocol for dried blood spots e g NucleoCards FTA cards Guthrie cards Before starting the preparation Check if Buffer B3 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 mr Pre lyse sample Add 180 pl 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 or use a shaking water bath Make sure that the samples are completely covered with lysis buffer during in cubation 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 1 MACHEREY NAGEL 01 2010 Rev 11 17 NucleoSpin Tissue 6 5 Support protocol for genomic DNA and viral DNA from blood samples B
35. ucleoSpin 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 MACHEREY NAGEL 01 2010 Rev 11 23 NucleoSpin Tissue 6 10 Support protocol for detection of Mycobacterium tuber culosis or Legionella pneumophila in sputum or bron choalveolar lavage Before starting the preparation Check if Buffer B3 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 NaOH 2 g NaOH 1 45 g sodium citrate 0 5 g N acetyl cystein Add water to 100 ml Prepare sample Add 200 500 pl sputum or bronchoalveolar lavage to an equal volume 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 provi
Download Pdf Manuals
Related Search