InviMag Tissue DNA Mini Kit/ KF96
Contents
1. Principle and procedure The InviMag Tissue DNA Mini Kit KF96 procedure comprises the following steps o Lysis of sample material and protein digestion o Binding the genomic DNA to the magnetic beads o Washing of the DNA and elimination of ethanol o Elution of DNA After lysis the DNA binds to the magnetic beads Contaminations and enzyme inhibitors are efficiently removed during the following three wash steps and highly purified DNA is eluted in Elution Buffer This manual contains 4 protocols pages 14 17 Lysis Samples are lysed at denaturing non chaotropic conditions using elevated temperatures in the presence of Lysis Buffer G and Proteinase K In case of a large sample number the preparation of a master mixture with a volume 5 greater than that required for the processing of all samples is recommended Mix the master mix carefully prior to use Binding of the DNA After adding Binding Buffer A and MAP Solution A to the lysate in a 2 0 ml Deep Well Plate the DNA is bound to the surface of the magnetic beads Removing residual contaminants Contaminants are efficiently removed using Wash Buffer while the DNA remains bound to the magnetic beads Elution The DNA is eluted from the beads using 150 ul Elution Buffer The eluted DNA is ready to use in different subsequent downstream applications such as PCR amplification genotyping restriction digestion and SNP analysis etc Yield and quality of genomic DNA The a2. 20 C can cause shearing of DNA particularly if the DNA is exposed to repeated freeze thaw cycles Plasmid DNA and other small circular DNAs can be stored at 2 8 C or at 20 C Drying dissolving and pipetting DNA Avoid overdrying genomic DNA after ethanol precipitation It is better to let it air dry than to use a vacuum although vacuum drying can be used with caution Plasmid DNA and other small circular DNAs can be vacuum dried To help dissolve the DNA carefully invert the tubes several times after adding buffer and tap the tube gently on the side Alternatively let the DNA stand in buffer overnight at 2 8 C Minimize vortexing of genomic DNA since this can cause shearing Avoid vigorous pipetting Pipetting genomic DNA through small tip openings causes shearing or nicking One way to decrease shearing of genomic DNA is to use special tips that have wide openings designed for pipetting genomic DNA Regular pipette tips pose no problem for plasmid DNA and other small 25 InviMag Tissue DNA Mini Kit KF96 0515 Ordering Information Product Package size Catalogue No InviMag Tissue DNA Mini Kit KF96 1 x 96 preparations 7432300100 InviMag Tissue DNA Mini Kit KF96 5 x 96 preparations 7432300200 Single components for InviMag Tissue DNA Mini Kit KF96 Lysis Buffer G 60 ml 7432301300 Binding Buffer A 30 ml 7432302700 MAP Solution A 1 ml 7432305100 Wash Buffer add 140 ml ethanol 60 ml 7432303200 Elution Buffer 30 ml 74323
3. Plates and Tip Plates are the same Use one provided Elution Plate as a Tip Plate 4 InviMag Tissue DNA Mini Kit KF96 0515 Symbols aall Manufacturer Eor Lot number Catalogue number Expiry date Consult operating instructions Temperature limitation ox API EE Do not reuse Storage All reagents and kit contents of the InviMag Tissue DNA Mini Kit KF96 except MAP Solution A are stable for at least 12 months MAP Solution A is stable for at least 6 months All buffers and kit contents of the InviMag Tissue DNA Mini Kit KF96 except dissolved Proteinase K and MAP Solution A should be stored at room temperature RT Proteinase K Dissolved Proteinase K must be stored at 20 C Dividing the Proteinase K into aliquots and storage at 20 C is recommended MAP Solution A The magnetic beads should be stored at 2 8 C Wash Buffers Binding Buffer Wash Buffers and Binding Buffers charged with either ethanol or isopropanol should be stored at room temperature and must be appropriately sealed If there are any precipitates visible within the provided solutions dissolve them by carefully warming up to room temperature up to 30 C 1 M DTT not provided Store the 1 M DTT solution at 20 C to prevent oxidative damage The DTT solution is stable for 12 months at this condition Room temperature RT is defined as range from 15 30 C Quality control and product warranty STRATEC Molecular warrants the corr
4. Tissue DNA Mini Kit KF96 cannot be excluded in all experimental setups If removal of RNA contaminations is required add 40 ul RNase A stock solution 10 mg ml before the addition of Binding Buffer A Vortex shortly and incubate the sample at room temperature for 5 min Then continue with the protocol description The elution can be done by using reduced volumes of Elution Buffer minimal 100 ul This may result in a higher DNA concentration but will reduce the total yield Please keep in mind that a change of volume has to be changed in the provided assay file too by using the Thermo Bindlt software InviMag Tissue DNA Mini Kit KF96 0515 Protocol 1 Isolation of genomic DNA from up to 20 mg tissue Please read the instructions carefully and conduct the prepared procedure Attention Please be aware that the Binding Buffer A has to be prepared see instructions page 12 I Sample Lysis A Sample lysis in a 1 5 ml reaction tube not provided 1 Cut the tissue sample app 0 5 cm of the mouse tail or up to 20 mg tissue the biopsy sample frozen section or insects into small pieces or homogenize the starting material 2 Transfer the tissue sample into a 1 5 ml reaction tube 3 Add 400 ul of Lysis Buffer G and 25 ul Proteinase K Important Vortex the tube for 5 s An incomplete mixing will reduce quality and yield of the isolated DNA 4 Incubate the sample at 56 C for app 2h or overnight at 52 C while continuously sh
5. amount of MAP Solution A too much Elution Buffer incorrect storage of starting material incorrect storage of starting material old material ethanol carryover during elution salt carry over during elution small part of the magnetic particles are left in the elution 23 Comments and suggestions increase lyses time reduce amount of starting material use a higher volume of Elution Buffer be sure you pipet the Elution Buffer with the correct amount to the correct position mix MAP Solution A thoroughly before pipetting to the Binding Plate elute the DNA with lower volume of Elution Buffer ensure that the storage of starting material was correctly Avoid thawing of the material ensure that the storage of starting material was correctly Avoid thawing of the material ensure that the starting material is fresh or stored under appropriate condition for long time storage at 20 C avoid thawing and freezing of the material old material often contains degraded DNA increase drying time for removing of ethanol check the Wash Buffers for salt precipitates If there are any precipitates visible solve them by carefully warming up to 30 C ensure that the Wash Buffers are at room temperature centrifuge at full soeed for 1 min and transfer supernatant to a new tube InviMag Tissue DNA Mini Kit KF96 0515 Appendix KingFisher Bindlt Software 3 2 or higher versions Bindlt software 3 2 or
6. gloves immediately Do not combine components of different kits unless the lot numbers are identical Avoid microbial contamination of the kit reagents To minimize the risk of infections from potentially infectious material we recommend working under laminar air flow until the samples are lysed o The kit should only be used by trained personnel O O O O O Preparing reagents and buffers Before starting a run bring all reagents to room temperature Where necessary gently mix and re dissolve any precipitates by warming up to 30 C Swirl gently to avoid foaming Lysis Buffer G and Elution Buffer are ready to use 1 x 96 DNA extractions Add 21 ml 99 7 Isopropanol to the Binding Buffer A Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times Dilute Proteinase K by addition of 1 ml of ddH2O mix thoroughly until completely dissolving and store at 20 C Add 105 ml of 96 100 ethanol to the bottle Wash Buffer mix thoroughly and keep the bottle always firmly closed 5 x 96 DNA extractions Add 84 ml 99 7 Isopropanol to the Binding Buffer A Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times Dilute Proteinase K by addition of 13 ml of ddH20 mix thoroughly until completely dissolving and store at 20 C Add 140 ml of 96 100 ethanol to the bottle Wash Buffer mix thoroughly and keep the bottle always firmly closed Reagen
7. the KF96 and KFflex96 instrument Reagent info Tip Plate Name Binding Plate Name Lysed sample Binding Buffer A Map Solution A Washing Plate 1 Name Wash Buffer Washing Plate 2 Name Wash Buffer Washing Plate 3 Name Wash Buffer Flution Plate Name Elution Buffer Dispensed reagents Well volume ul Well volume ul 425 200 20 Well volume ul 800 Well volume ul 800 Well volume ul 800 Well volume ul 150 King Fisher 96 KF plate Total reagent volume pl Microtiter DW 96 plate Total reagent volume ul Microtiter DW 96 plate Total reagent volume ul Microtiter DW 96 plate Total reagent volume ul Microtiter DW 96 plate Total reagent volume ul King Fisher 96 KF plate Total reagent volume ul Type Type Sample Reagent Reagent Type Reagent Type Reagent Type Reagent Type Reagent The protocol does not contain dispensed reagents 21 InviMag Tissue DNA Mini Kit KF96 0515 Steps data Tip p Pick Up Binding Beginning of step Mixing heating End of step Washl Beginning of step Mixing heating End of step Wash2 Beginning of step Mixing heating End of step Wash3 Beginning of step Mixing heating End of step Drying Elution Beginning of step Mixing heating End of step Bead Removal Leave 96 DW tip comb Tip Plate Binding Plate Precollect Release time speed Mixing t
8. with a KingFisher instrument please carefully read the manufacturer s manual 1 Switch on the KF96 KFflex96 instrument Note Use one provided Elution Plate as Tip Plate These are identical 2 Prefill the Deep Well Plates with the corresponding buffers during the lysis Note Please avoid evaporation of the prefilled buffer components by sealing the Deep Well Plates with a sealing foil or with parafilm Important Mix the bottle with the MAP Solution A by vigorously vortexing Tip Plate Place the Tip Plate with a KF 96 Tip Comb for DW magnets on an Elution Plate Binding Plate Transfer the cleared lysate carefully into the cavity of a 2 ml DWP prefilled with 200 ul Binding Buffer A and 20 ul MAP Solution A Washing Plate 1 Pipet 800 ul Wash Buffer into a 2 ml Deep Well Plate Washing Plate 2 Pipet 800 ul Wash Buffer into a 2 ml Deep Well Plate Washing Plate 3 Pipet 800 ul Wash Buffer into a 2 ml Deep Well Plate Elution Plate Pipet 150 ul Elution Buffer into the KF Elution Plate 3 Choose the program InviMag Tissue DNA KF96 or InviMag Tissue DNA KFflex96 depending on the used instrument press the START button 4 Insert the prefilled Plates onto the right position of instrument surface by following the specification shown on the display and confirm every loading step by pressing the START button 5 When all prefilled plates are loaded into the machine press the START button finally to begin the run I
9. 04000 KingFisher 96 and consumables KingFisher 96 Magnetic Particle Processor 100 240V 50 60Hz 5400500 including one magnetic head KingFisher 96 Head for Deep Well plate 24073430 KingFisher 96 tip comb for PCR magnets 8 x 10 pcs box 97002514 KingFisher 96 tip comb for KF magnets 10 x 10 pcs box 97002524 KingFisher 96 tip comb for DW magnets 10 x 10 pcs box 97002534 KingFisher 96 KF plate 200ul 48 plates box 97002540 Microtiter deep well 96 plate 50 plates box 95040450 Related Products Package size Catalogue No Invisorb Spin Tissue Mini Kit 50 preparations 1032100200 Invisorb Spin Tissue Mini Kit 250 preparations 1032100300 Invisorb DNA Tissue HTS 96 Kit C 4 x 96 preparations 7032300300 Invisorb DNA Tissue HTS 96 Kit C 24 x 96 preparations 7032300400 Possible suppliers for lsopropanol Carl Roth Applichem Fa Sigma 2 Propanol 2 Propanol fur die Molekularbiologie 2 Propanol Rotipuran gt 99 7 p a ACS ISO Order No A3928 Order No 59304 1L F Order No 6752 26 InviMag Tissue DNA Mini Kit KF96 0515 Stratecee molecular STRATEC Molecular GmbH Robert Rossle Str 10 13125 Berlin Germany Phone 49 30 94 89 29 01 Fax 49 30 94 89 29 09 E mail info berlin stratec com www stratec com 1G3b02 05 2015
10. A Mini Kit KF96 3 Kit contents of InviMag Tissue DNA Mini Kit KF96 w o plastic 4 Symbols 5 Storage 5 Quality control and product warranty 5 Intended use 6 Product use limitation 6 Safety information 7 Product characteristic of the InviMag Tissue DNA Mini Kit KF96 8 Sampling and sample storage of the starting material 9 Principle and procedure 10 Yield and quality of genomic DNA 10 Scheme of the InviMag Tissue DNA Mini Kit KF96 11 Important notes 12 Preparing reagents and buffers 12 Reagents and equipment to be supplied by user 12 Important indications for the InviMag Tissue DNA Mini Kit KF96 14 Protocol 1 Isolation of genomic DNA from up to 20 mg tissue 14 Protocol 2 Isolation of genomic DNA from paraffin embedded tissue 15 Protocol 3 Isolation of genomic DNA from formalin fixed tissue or formalin fixed paraffin embedded tissue FFPE 16 Protocol 4 Isolation and purification of DNA from eucaryotic cells 17 Starting a run on a KF96 KFflex96 instrument 19 For self programming of the KF96 and KFflex96 instrument 21 Troubleshooting 23 Appendix 24 General notes on handling DNA 25 Ordering Information 26 2 InviMag Tissue DNA Mini Kit KF96 0515 Kit contents of InviMag Tissue DNA Mini Kit KF96 Store the MAP Solution A at 2 8 C Store dissolved Proteinase K at 20 C Store all other kit components at room temperature RT 1 x 96 extractions 5 x 96 extractions 7432300100 7432300200 50 ml 220 ml 9 ml 36 ml
11. aking e g by using a thermomixer 5 During lysis prefill all plates with required buffers and appropriate volumes see Starting a Run page 19 6 After lysis centrifuge the sample for 2 min at max speed and transfer the cleared supernatant into the cavity of the Binding Plate prefilled with 200 ul Binding Buffer A and 20 ul MAP Solution A see Starting a Run page 19 B Sample lysis in a Deep Well Plate not provided 1 Cut the tissue app 0 5 cm of the mouse tail or up to 20 mg tissue into small pieces 2 Transfer the tissue sample into the cavities of the Deep Well Plate refers to Binding Plate and add 400 ul Lysis Buffer G and 25 ul Proteinase K 3 Seal the Deep Well Plate with a Sealing Foil Important Note Vortex the Deep Well Plate 10 times An incomplete mixing will reduce quality and yield of the isolated DNA 4 Incubate the Deep Well Plate at 56 C for 2 h or overnight at 52 C use e g hybridization oven or incubator until lysis is complete Important Note Vortex the Deep Well Plate each 20 minutes for 20 sec Vortexing is essentially for an efficient lysis step No vortexing will lead to a dramatic reduction of the final yield 5 During lysis prefill all plates with the required buffers and appropriate volumes see Starting a Run page 19 6 After lysis centrifuge the Deep Well Plate with the lysed samples for 10 min at 4 000 rom to spin down the unlysed material 7 Trans
12. ample and remove the supernatant very carefully 2 Add 1 ml PBS Buffer to the tissue sample and vortex carefully Centrifuge for 1 min at maximum speed of 13 000 x g 12000 rpm to pellet the tissue sample Discard the supernatant very carefully Important Mechanical grinding or a cutting of the material is recommended before or during lysis 3 Continue with the lysis step 1 of protocol 1 page 14 and add DTT to a final concentration of 1 mM DTT 1 ul of 1 M DTT per ml of Lysis Buffer G to the Lysis Buffer G Incubate the reaction tube at 56 C until the lysis is complete while continuously shaking Lysis time should be at least 2 h 4 During lysis prefill all plates with the required buffers and appropriate volumes see Starting a Run page 19 16 InviMag Tissue DNA Mini Kit KF96 0515 5 After lysis centrifuge the sample for 2 min at max speed and transfer the cleared supernatant carefully into the cavity of the Binding Plate prefilled with 200 ul Binding Buffer A and 20 ul MAP Solution A see Starting a Run page 19 Protocol 4 solation and purification of DNA from eucaryotic cells Please read the instructions carefully and conduct the prepared procedure Attention Please be aware that the Binding Buffer A has to be prepared see instructions page 12 Harvesting cells Cells grown in suspension Spin up to 1 x 10 cells for 5 min at 300 x g 1500 rpm Discard the supernatant and carefully remove all med
13. down for 5 min at 300 x g 1500 rpm Discard the supernatant and remove all media completely Do not disturb the cell pellet Add 400 ul Lysis Buffer G and 25 ul Proteinase K Seal the Deep Well Plate with a Sealing Foil os ce A ae 17 InviMag Tissue DNA Mini Kit KF96 0515 Important Note Vortex the Deep Well Plate 10 times An incomplete mixing will reduce quality and yield of the isolated DNA 6 Incubate the Deep Well Plate at 56 C for 2 h e g use a hybridization oven or incubator until lysis is complete Important Note Vortex the Deep Well Plate each 20 minutes for 20 sec Vortexing is essentially for an efficient lysis step No vortexing will lead to a significant reduction of the final yield 7 During lysis prefill all plates with the required buffers and appropriate volumes see Starting a Run page 19 8 After lysis centrifuge the Deep Well Plate with the lysed samples for 10 min at 1700 x g 4000 rpm to spin down unlysed material 9 Carefully transfer the cleared supernatant from the lysate into the cavity of the Binding Plate prefilled with 200 ul Binding Buffer A and 20 ul MAP Solution A see Starting a Run page 19 InviMag Tissue DNA Mini Kit KF96 0515 Starting a run on a KF96 KFflex96 instrument Preliminary Steps to process the sample onto the KingFisher System Attention Please be aware that the Binding Buffer A has to be prepared see instructions page 12 Important Before working
14. e 1 for KF96 KFflex96 KFDuo connection Pointing device Mouse or equivalent is required CD ROM drive XVGA monitor with at least 1024x768 resolution and at least a 16 bit color Monitor color settings i onitor color setting environment If the actual Windows Service Packs are not installed on the corresponding lab computer they can be downloaded from the Microsoft web pages http www microsoft com oc InviMag Tissue DNA Mini Kit KF96 0515 General notes on handling DNA Nature of DNA The length and delicate physical nature of DNA require careful handling to avoid damage due to shearing and enzymatic degradation Other conditions that affect the integrity and stability of DNA include acidic and alkaline environments high temperature and UV irradiation Careful isolation and handling of high molecular weight DNA is necessary to ensure it will function well in various downstream applications Damaged DNA could perform poorly in applications such as genomic Southern blotting long template PCR and construction of cosmid libraries Handling fresh and stored material before the extraction of DNA For the isolation of genomic DNA from cells or tissues use either fresh samples or samples that have been quickly frozen in liquid nitrogen and stored at 70 C This procedure minimizes degradation of crude DNA by limiting the activity of endogenous nucleases Storage of DNA Store genomic DNA at 2 8 C Storing genomic DNA at
15. e by grinding with mortar and pestle using liquid nitrogen due to their stable chitin coat The sample can be stored for a short time at 2 8 C in Lysis Buffer G Paraffin slices formalin fixed tissue FFPE These samples can be stored at room temperature 18 25 C By appropriate paraffin embedding or formalin fixation pure DNA can be isolated from this starting material but paratfin embedding or formalin fixation leads to a reduced DNA quality An improper contact of the tissue with formalin will dramatically reduce the DNA yield Cells grown in suspension Spin up to 1x10 cells for 5 min at 300 x g 1 500 rpm Discard the supernatant and remove all media completely Do not disturb the cell pellet At this point the cells may be frozen at 80 C 20 C for future or immediately use 9 InviMag Tissue DNA Mini Kit KF96 0515 Cells grown in a monolayer Aspirate the media completely from the cells and continue immediately with the lysis step Alternatively the cells can be detached by trypsination e g cultivation in larger culture vessels like dishes gt 35 mm flasks gt 12 5 cm Transfer the cells to a 50 ml reaction tube pellet by centrifugation at 300 x g 1 500 rpm for 5 min and aspirate supernatant completely STRATEC Molecular will be released of its responsibilities if other sample materials than described in the Intended Use are processed or if the sample preparation protocols are changed or modified
16. e to the high purity the isolated DNA is ready to use for in vitro diagnostic analysis and for a broad panel of downstream applications or can be stored at 20 C for subsequent use The kit is neither suitable for isolation of DNA from blood stool sample swabs dried blood stains or cell free body fluids like cerebrospinal fluid synovial fluid and urine nor from bacteria plant material fungi parasites or the purification of RNA The application of the kit for isolation and purification of viral DNA has not been evaluated C 3 Compliance with EU Directive 98 79 EC on in vitro medical devices Not for in vitro diagnostic use in countries where the EU Directive 98 79 EC on in vitro medical devices is not recognized Trademarks InviMag Invisorb Registered marks trademarks etc used in this document even when not specifically marked as such are not to be considered unprotected by law The Invisorb technology is covered by patents and patent applications US 6 110363 US 6 043 354 US 6 037 465 EP 0880535 WO 9728171 WO 9534569 EP 0765335 DE 19506887 DE 10041825 2 WO 0034463 InviMag and Invisorb are registered trademarks of STRATEC Biomedical AG The PCR process is covered by US Patents 4 683 195 and 4 683 202 and foreign equivalents owned by Hoffmann La Roche AG 2015 STRATEC Molecular all rights reserved InviMag Tissue DNA Mini Kit KF96 0515 Table of Content Kit contents of InviMag Tissue DN
17. ect function of the InviMag Tissue DNA Mini Kit KF96 for applications as described in this manual Purchasers must determine the suitability of the product for its particular use Should any product fail to perform in the applications described in the manual STRATEC Molecular will check the lot and if a problem is approved STRATEC Molecular will replace the product free of charge STRATEC Molecular reserves the right to change alter or modify any product to enhance its performance and design at any time In accordance with STRATEC Molecular s ISO 9001 2000 and ISO EN 13485 certified Quality Management System the performance of all components of the InviMag Tissue DNA Mini Kit KF96 is tested separately against predetermined specifications routinely on lot to lot to ensure consistent product quality If you have any questions or problems regarding any aspects of InviMag Tissue DNA Mini Kit KF96 or other STRATEC Molecular products please do not hesitate to contact us A copy of STRATEC Molecular s terms and conditions can be obtained upon request or are presented at the STRATEC Molecular webpage 5 InviMag Tissue DNA Mini Kit KF96 0515 For technical support or further information please contact from Germany 49 0 30 9489 2901 2910 from abroad 49 0 30 9489 2907 or contact your local distributor Intended use The InviMag Tissue DNA Mini Kit KF96 is designed for semi automated extraction and purification of total gen
18. f nucleic acids with coated magnetic particles at adapted buffer conditions The KingFisher instrument performs all steps of the DNA purification procedure automatically without any user intervention except the external lysis step Due to the different possible starting materials which require different starting protocols an automated lysis step is not possible However the procedure requires only minimal interaction by the user Sample cross contamination and reagent cross over is effectively eliminated by this automated purification process The KingFisher instrument uses magnetic rods to transport the DNA binding magnetic particles through the various purification phases binding washing and elution The buffer volumes and other liquids required for DNA isolation is reduced to a minimum After a sample specific cell lysis using Lysis Buffer G and Proteinase K optimal binding conditions are adjusted upon addition of Binding Buffer A The DNA bound to the simultaneously added magnetic particles is then separated from solution by the magnetic rods controlled by the KingFisher instrument Subsequent to three washing steps of the particle bound nucleic acids the DNA is eluted in Elution Buffer Due to the high purity the eluted total genomic and mitochondrial DNA is ready to use for a broad panel of downstream applications like PCR Genotyping Restriction digestion SNP Analysis Specially optimized lysis conditions allow an efficien
19. fer into the KF Elution Plate same size as Tip Plate Mix the starting material with 400 ul Lysis Buffer G and 25 ul Proteinase K and incubate at 56 C for 2 h Addition of 200 ul Binding Buffer A follow preparing instructions page 12 and 20 ul MAP Solution A to the lysate DNA binds to magnetic particles Magnetic separation Washing of the particle fixed genomic DNA Magnetic separation Elution of genomic DNA Magnetic Separation Pure DNA Elution Plates and Tip Plates are the same Use one provided Elution Plate as a Tip Plate InviMag Tissue DNA Mini Kit KF96 0515 Important notes Important points before starting a protocol After receiving the kit inspect the product and its components as well as the package for any visible damages and correct quantities If there are any unconformities immediately notify STRATEC Molecular If buffer bottles are damaged contact the STRATEC Molecular Technical Services or your local distributor In case of liquid spillage refer to Safety Information see page 7 Do not use damaged kit components because their use may lead to poor kit performance o Always change pipet tips between liquid transfers To avoid cross contamination we recommend the use of aerosol barrier pipet tips o All centrifugation steps are carried out at room temperature When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles Discard contaminated
20. fer the cleared supernatant of the lysate carefully into the cavity of the Binding Plate prefilled with 200 ul Binding Buffer A and 20 ul MAP Solution A see Starting a Run page 19 InviMag Tissue DNA Mini Kit KF96 0515 Protocol 2 Isolation of genomic DNA from paraffin embedded tissue Please read the instructions carefully and conduct the prepared procedure Attention Please be aware that the Binding Buffer A has to be prepared see instructions page 12 De paraffination Transfer the starting material into a 1 5 ml reaction tube not provided Add 1 ml Octane not provided and vortex carefully to dissolve the paraffin Follow the dissolution until the tissue sample looks transparent solid paraffin is still white Centrifuge for 2 min at maximum speed to pellet down the tissue sample Discard the supernatant very carefully This step should be repeated if paraffin residues remain in the sample Add 0 5 ml of 96 100 ethanol to the tissue sample and mix the tube thoroughly Centrifuge shortly and remove the ethanol by aspiration Then incubate the opened reaction tube at 52 C to evaporate the residual ethanol A mechanical grinding or a cutting of the deparatfined material will increase the lysis efficiency I Sample Lysis A Sample lysis in a 1 5 ml reaction tube not provided 1 Transfer the starting material after deparaffination see above into a 1 5 ml reaction tube and add 400 ul of Lysis Buffer G and 25
21. final volume 30 ml final volume 120 ml 2x 1 2 ml 10 5 ml 2X 45 ml 6 x 60 ml final volume 2 x 150 ml final volume 6 x 200 ml 30 ml 120 ml Catalogue No Lysis Buffer G Binding Buffer A Proteinase K working solution MAP Solution A Wash Buffer Elution Buffer 2 0 ml Deep Well Plate KF 96 Tip Comb for DW magnets 200 ul Elution Plate 1 5 ml Receiver 2x50 Sealing Foils c o D Initial steps Add 21 ml of 99 7 Isopropanol to the Binding Buffer A Mix by intensive shaking by inverting for 1 min Add 84 ml of 99 7 Isopropanol to the Binding Buffer A Mix by intensive shaking by inverting for 1 min Dilute Proteinase K by addition of 1 ml of ddH20 mix thoroughly until completely dissolving and store at Dilute Proteinase K by addition of 13 ml of ddH2O mix thoroughly until completely dissolving and store at 20 C Add 140 ml of 96 100 ethanol to the bottle Wash Buffer mix thoroughly and keep the bottle always firmly closed Divide the MAP Solution into aliquots according your need 20 C Add 105 ml of 96 100 ethanol to the bottle Wash Buffer mix thoroughly and keep the bottle always firmly closed Elution Plates and Tip Plates are the same Use one provided Elution Plate as a Tip Plate 3 InviMag Tissue DNA Mini Kit KF96 0515 Kit contents of InviMag Tissue DNA Mini Kit KF96 w o plastic Store the MAP Solution A at 2 8 C Store dissolved Prote
22. higher versions were and may be used to create assay files for the KFmL KF96 KFflex96 or KF Duo instruments The provided assay file s can either be transferred onto the corresponding workstation s or be started directly from within the Bindlt software after assay import Please keep in mind that assay s run from within the Bindlt software are not stored in the workstation memory Important Be advised that Bindlt SW 3 2 or higher versions use a new unique file extension Therefore it is not possible to import assay files created with Bindlt 3 2 or higher versions into older Bindlt software versions Please ask your local Thermo Scientific distributor for a software update Note When creating assay files for usage with KingFisher instruments in combination with Microtiter Deep Well plates e g Thermo Electron it is essential to use the KingFisher software 3 2 or higher versions for assay development because this software version includes the correct adjustments for the microtiter plate It is highly recommended to use Thermo Microtiter Deep Well plates with KF96 KFflex96 KF Duo workstations to ensure the best purification result Minimum system requirements for Bindlt Software 3 2 or higher versions PC requirements nas SPE MS Windows XP Pro with SP3 Windows Vista SP2 Windows 7 Disk space 500 MB free disk space Processor Intel Pentium gt 1 GHz Memory 1 GB RAM Serial ports available 1 for KFmL connection USB ports availabl
23. ia Cells grown ina monolayer In large culture vessels dishes gt 35 mm flasks gt 12 5 cm detach cells by trypsination Transfer the cells to a centrifuge tube and sediment the cell by centrifugation at 1500 rom for 5 min Remove the supernatant completely In small culture vessels 96 24 12 6 well plates 35 mm dishes 12 5 cm flasks discard the media completely and continue with the lysis immediately I Sample Lysis A Sample lysis in a 1 5 ml reaction tube not provided 1 Transfer up to 1x10 cells into a 1 5 ml reaction tube 2 Spin down for 5 min at 300 x g 1500 rpm 3 Discard the supernatant and remove all media completely Do not disturb the cell pellet 4 Add 400 ul of Lysis Buffer G and 25 ul Proteinase K Important Vortex the tube for 5 s An incomplete mixing will reduce the quality and yield of the isolated DNA 5 Incubate the sample at 56 C for 2h while continuously shaking e g by using a thermomixer During lysis prefill all plates with the required buffers and appropriate volumes see Starting a Run page 19 N After lysis centrifuge the sample for 2 min at max speed and transfer the cleared supernatant carefully into the cavity of the Binding Plate prefilled with 200 ul Binding Buffer A and 20 ul MAP Solution A see Starting a Run page 19 U Sample lysis in a Deep Well Plate Transfer up to 1x10 cells into the cavity of a Deep Well Plate Spin
24. ime speed Heating during mixing Postmix Collect count Collect time s Washing Plate 1 Precollect Release time speed Mixing time speed Heating during mixing Postmix Collect count Collect time s Washing Plate 2 Precollect Release time speed Mixing time speed Heating during mixing Postmix Collect count Collect time s Washing Plate 3 Precollect Release time speed Mixing time speed Heating during mixing Postmix Collect count Collect time s Washing Plate 3 Dry time Tip position Elution Plate Precollect Release time speed Mixing time speed Heating temperature C Preheat Postmix Collect count Collect time s Washing Plate 3 Release time speed Tip Plate No 00 00 10 Fast 00 05 00 Medium No No 4 5 No 00 00 30 Fast 00 01 30 Fast No No 3 5 No 00 00 30 Medium 00 01 30 Fast No No 3 5 No 00 00 30 Medium 00 01 30 Fast 00 05 00 Outside well tube No 00 00 10 Medium 00 10 00 Slow 70 Yes o 7 E Ut Z 00 00 30 Fast 09 InviMag Tissue DNA Mini Kit KF96 0515 Troubleshooting Problem low amount of extracted DNA low concentration of extracted DNA degraded or sheared DNA DNA does not perform well in downstream applications e g real time PCR or PCR low Aa260 A02g80 ratio from UV measurement eluted DNA is brown colored Probable cause insufficient lysis incomplete elution low
25. inase K at 20 C Store all other kit components at room temperature RT 1 x 96 extractions 5 x 96 extractions 7432300150 7432300250 50 ml 220 ml 9 ml 36 ml final volume 30 ml final volume 120 ml 2x 1 2 ml 10 5 ml 2 x 45 ml 6 x 60 ml final volume 2 x 150 ml final volume 6 x 200 ml 30 ml Catalogue No Lysis Buffer G Binding Buffer A Proteinase K working solution MAP Solution A Wash Buffer Elution Buffer 1 5 ml Receiver Tubes 2x50 Sealing Foils Initial steps Add 21 ml of 99 7 isopropanol to the Binding Buffer A Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times Add 84 ml 99 7 isopropanol to the Binding Buffer A Mix by intensive shaking by inverting for 1 min shortly before use mix by inverting several times Dilute Proteinase K by addition of 1 ml of ddH2O mix thoroughly Dilute Proteinase K by addition of 13 ml of ddH2O mix thoroughly until completely dissolving and store at 20 C until completely dissolving and store at 20 C Add 105 ml of 96 100 ethanol to the bottle Wash Buffer mix thoroughly and keep the bottle always firmly closed Add 140 ml of 96 100 ethanol to the bottle Wash Buffer mix thoroughly and keep the bottle always firmly closed Divide the MAP Solution into aliquots according your need Plastic to be supplied by user see order information KF 96 Tip Comb for DW i 5 magnets Elution
26. ll The following extraction steps run automatically on the KingFisher System Binding of the DNA Automatically sample mixing for 5 min MAP separation Moving of the MAP into Washing Plate 1 First Washing Automatically sample mixing for 90 s MAP separation Moving of the MAP into the Washing Plate 2 Second Washing Automatically sample mixing for 90 s MAP separation Moving of the MAP into Washing Plate 3 19 InviMag Tissue DNA Mini Kit KF96 0515 Third Washing and Drying Automatically sample mixing for 90s MAP separation Air drying of the MAP outsight Washing Plate 3 for 5 min Moving of the MAP into the Elution Plate Elution of the DNA Incubation of the MAP into the Elution Plate for 10 min by mixing at 70 C MAP separation The MAP will then be removed into the Washing Plate 3 disposal The extracted DNA can now be transferred into new 1 5 ml Receiver Tubes Important Notes After finishing the extraction protocol the Elution Plate contains the extracted genomic DNA Store the DNA under adequate conditions We recommend to transfer the extracted DNA into 1 5 ml Receiver Tubes for further storage and freeze the DNA at 20 If the extracted DNA contains carryover of magnetic particle transfer the DNA to 1 5 ml reaction tube centrifuge at maximum speed 13000 rpm for 1 min and transfer the DNA containing supernatant into a receiver tube 20 InviMag Tissue DNA Mini Kit KF96 0515 For self programming of
27. mount of purified DNA in the InviMag Tissue DNA Mini Kit KF96 procedure from tissue or tissue related samples depends on the sample source transport conditions storage and age of the sample Yield and quality of isolated genomic DNA is suitable for any molecular diagnostic detection system The diagnostic tests should be performed accordingly to manufacturers specifications 10 InviMag Tissue DNA Mini Kit KF96 0515 Scheme of the InviMag Tissue DNA Mini Kit KF96 Please read protocols prior the start of the preparation carefully Transfer the sample into a 1 5 ml reaction tube not provided or into a cavity of a 2 0 ml Deep Well Plate not provided Add 400 ul Lysis Buffer G add 25 ul Proteinase K to the sample and mix by vortexing Incubate for 2 h at 56 C or overnight at 52 C while continuously shaking Spin down unlysed material Transfer each sample into a well of the 2 0 ml Deep Well Plate refers to Binding Plate Prefill all plates of the KingFisher 96 with required buffers and the appropriate volumes Tip Plate Binding Plate Washing Plate_1 Washing Plate_2 Washing Plate_3 Elution Plate Place in the KF 96 Tip Comb for DW magnets on a KF Elution Plate Add 200 ul Binding Buffer A and 20 ul MAP Solution A to the lysate Add 800 ul Wash Buffer into a 2 ml Deep Well Plate Add 800 ul Wash Buffer into a 2 ml Deep Well Plate Add 800 ul Wash Buffer into a 2 ml Deep Well Plate Add 150 ul Elution Buf
28. mperature RT for 2 3 hours For short time storage up to one week samples may be stored at 2 8 C For long term storage we recommend freezing samples at 20 C or 80 C Multiple thawing and freezing cycles before isolating the DNA should be avoided because this can degrade the DNA Biopsy material frozen section Best results are obtained with fresh material or material that has been immediately frozen and stored at 20 C or 80 C Repeated freezing and thawing cycles of stored samples should be avoided because this can lead to a degraded DNA Use of poor quality Starting material influences the yield of purified DNA The amount of purified DNA in the InviMag Tissue DNA Mini Kit KFmL procedure using 5 20 mg tissue sample depends on kind of starting material The thawing process can be performed directly in Lysis Buffer G Rodent tail Best results are obtained with fresh material or material that has been immediately frozen and stored at 20 C or 80 C Repeated freezing and thawing cycles of stored samples should be avoided because this can lead to a reduced DNA size A prolonged lysis time may lead to degradation of DNA too Crushing the rodent tails reduces the overall lysis time An overnight lysis will probably shear the DNA but is possible Insects Best results are obtained with fresh material or material that has been immediately frozen and stored at 20 C or 80 C Insects must be homogenized before lysis for exampl
29. must be stored in the laboratory and must not be used for other purposes than intended The kit and its contents are unfit for consumption 6 InviMag Tissue DNA Mini Kit KF96 0515 Safety information When and while working with chemicals always wear a suitable lab coat disposable gloves and protective goggles Avoid direct skin contact Adhere to the legal requirements for working with biological material For more information please consult the appropriate material safety data sheets MSDS These are available online in convenient and compact PDF format at www stratec com for each STRATEC Molecular kit and its components If buffer bottles are damaged or leaking WEAR GLOVES AND PROTECTIVE GOGGLES when discarding the bottles in order to avoid any injuries STRATEC Molecular has not tested the liquid waste generated by the InviMag Tissue DNA Mini Kit KF96 procedures for residual infectious materials Contamination of the liquid waste with residual infectious materials is highly unlikely but cannot be excluded completely Therefore liquid waste has to be considered infectious and be handled and discarded accordingly to local safety regulations European Community risk and safety phrases for the components of the InviMag Tissue DNA Mini Kit KF96 to which they apply are listed below as follows Proteinase K He danger H315 319 334 335 P280 305 351 338 310 405 H315 Causes skin irritation H319 Causes serious eye ir
30. omic and mitochondrial DNA from up to 96 tissue or tissue related samples using magnetic beads and the KF96 KFflex96 instrument The provided nucleic acid isolation protocols are suitable for semi automated preparation of DNA from fresh or frozen tissue samples biopsy samples frozen sections rodent tails insects paraffin embedded tissues formalin fixed tissues and eucaryotic cell pellets For reproducible and high yields an appropriate sample storage is essential see Sampling and storage of the starting material page 9 All utilities reagents and plastic ware necessary for preparation of total DNA are provided by the InviMag Tissue DNA Mini Kit KF96 in different package sizes The procedures of the InviMag Tissue DNA Mini Kit KF96 have been optimized for the isolation of total DNA from up to 20 mg tissue sample app 0 5 cm rodent tail or 1 x 10 eucaryotic cells THE PRODUCT IS INTENDED FOR USE BY PROFESSIONALS SUCH AS TECHNICIANS PHYSICIANS AND BIOLOGISTS TRAINED IN MOLECULAR BIOLOGICAL TECHNIQUES It is designed to be used with any downstream application employing enzymatic amplification or other enzymatic modifications of DNA followed by signal detection or amplification Any diagnostic results generated by using the sample preparation procedure in conjunction with any downstream diagnostic assay should be interpreted with regard to other clinical or laboratory findings To minimize irregularities in diagnostic result
31. rial Standard formalin fixation and paraffin embedding procedures always result in significant fragmentation of nucleic acids To limit the extent of DNA fragmentation ensure that o Fix tissue samples in 4 10 formalin as quickly as possible after surgical removal o Use a fixation time of 14 24 h longer fixation leads to more DNA fragmentation resulting in poor performance in downstream assays o Thoroughly dehydrate samples before embedding residual formalin can inhibit the Proteinase K digest Formalin fixation and paraffin embedding procedures according to standard protocols always cause a degradation of nucleic acids The use of freshly cut sections with a thickness of up to 10 um is recommended Do not apply more than 8 sections with a surface area of 250 mm in one purification run Tissues with a high DNA content e g thymus request the use of fewer sections per preparation to avoid overloading the system For efficient purification of RNA from formalin fixed tissues we recommend the use of the Invisorb Tissue RNA Mini Kit which is optimized for high yields of usable RNA from these samples see Order Information page 26 2 Pretreatment 1 Transfer the formalin fixed starting material into a 1 5 ml reaction tube not provided Add 1 ml PBS Buffer containing 2mM DTT 2 ul of 1M DTT and agitate continuously for 20 min at 99 C Centrifuge for 1 min at maximum speed 13 000 x g 12000 rpm to pellet the tissue s
32. ritation H334 May cause allergy or asthma symptoms or breathing difficulties if inhaled H335 May cause respiratory irritation P280 Wear protective gloves protective clothing eye protection face protection P305 P351 P338 If in eyes Rinse cautiously with water for several minutes If present remove contact lenses and continue rinsing P310 Immediately call a POISON CENTER or doctor physician P405 Store locked up No toxic or hazardous chemicals like chaotropic components are used Emergency medical information can be obtained 24 hours a day from infotrac outside of USA 1 352 323 3500 inside of USA 1 800 535 5053 7 InviMag Tissue DNA Mini Kit KF96 0515 Product characteristic of the InviMag Tissue DNA Mini Kit KF96 The InviMag Tissue DNA Mini Kit KF96 procedure is the ideal tool for an efficient DNA extraction and purification from fresh or frozen tissue samples biopsy samples frozen sections rodent tails insects paraffin embedded tissues formalin fixed tissues and eucaryotic cell pellets in a convenient 96 well format using magnetic beads and the KF96 KFflex96 workstation Starting material Time for preparation up to 20 mg tissue samples 10 25 ug depends on app 35 min 0 5 cm rodent tail the starting material without lysis paraffin embedded tissue formalin fixed samples up to 1x10 mammalian cells The DNA isolation process is based on the interaction o
33. s adequate controls for downstream applications should be used The kit is in compliance with EU Directive 98 79 EC on in vitro medical devices But it is not for in vitro diagnostic use in countries where the EU Directive 98 79 EC on in vitro medical devices is not recognized Product use limitation The kit is neither validated for the isolation of DNA from stool samples whole blood bacteria fungi or viruses nor for isolation and purification of RNA The included chemicals are only useable once If the flow trace or starting material is changed no issue in operability will be given The user is responsible to validate the performance of the STRATEC Molecular product for any particular use STRATEC Molecular does not provide for validation of performance characteristics of the product with respect to specific applications STRATEC Molecular products may be used e g in clinical diagnostic laboratory systems conditioned upon the complete diagnostic system of the laboratory the laboratory has been validated pursuant to CLIA 88 regulations in the U S or equivalents in other countries All products sold by STRATEC Molecular are subject to extensive quality control procedures according to ISO 9001 2000 and ISO EN 13485 and are warranted to perform as described herein Any problems incidents or defects shall be reported to STRATEC Molecular immediately upon their detection The reagents and plastic parts are for laboratory use only They
34. stratecee molecular User manual InviM g Tissue DNA Mini Kit KF96 for use on KingFisher 96 and KingFisher Flex Thermo Fisher for automated purification of genomic DNA from up to 20 mg of tissue sample mouse or rodent tail paraffin embedded tissue formalin fixed tissue and eucaryotic cell pellets with magnetic beads 7432300X00 gaai STRATEC Molecular GmbH D 13125 Berlin Instruction for InviMag Tissue DNA Mini Kit KF96 The InviMag Tissue DNA Mini Kit KF96 combines the advantages of the innovative Invisorb technology with easy handling of magnetic particles for a very efficient and reliable isolation of nucleic acids with a high purity The DNA binding magnetic particles are characterized by a high surface area a uniform size distribution and good suspension stability and are therefore highly suitable for high throughput processing The InviMag Tissue DNA Mini Kit KF96 for isolation and purification of total genomic and mitochondrial DNA from tissue samples biopsy samples frozen sections rodent tails insects paraffin embedded tissue formalin fixed tissue FFPE and eucaryotic cell pellets in a 96 well format is designed for an optimal use on the KingFisher 96 or Flex workstation from Thermo Scientific The interplay of the DNA extraction and purification chemistry provided by the InviMag Tissue DNA Mini Kit KF96 with the KingFisher machine was intensely tested and validated Du
35. t purification of genomic DNA from paraffin embedded and formalin fixed tissue sections after a de paraffination step or a special treatment of the formalin fixed tissue without the need for overnight incubation O O O O Incubation at elevated temperatures before Proteinase K digestion in presence of DIT partially removes formalin cross linking of the released DNA improving yield as well as performance in downstream assays The InviMag Tissue DNA Mini Kit KF96 is supplied with a comprehensive manual describing four protocols page 14 17 for DNA purification from different sources The PCR process is covered by US Patents 4 683 195 and 4 683 202 and foreign equivalents owned by Hoffmann La Roche AG g InviMag Tissue DNA Mini Kit KF96 0515 For the isolation of DNA from a single tissue sample STRATEC Molecular offers the Invisorb Spin Tissue Mini Kit or for 8 96 samples the Invisorb DNA Tissue Mini HTS 96 Kit C for use in a centrifuge see Ordering Information page 26 For further information please contact 49 0 30 9489 2901 2910 in Germany and from foreign countries 49 0 30 9489 2907 or call your local distributor Sampling and sample storage of the starting material For reproducible and high yields appropriate sample storage is essential Yields may be varying from sample to sample depending on factors such as sample age kind of sample and storage conditions Tissue samples can be stored at room te
36. th the lysed samples for 10 min at 4000 rom to spin down the unlysed material 7 Transfer the cleared supernatant of the lysate carefully into the cavity of the Binding Plate prefilled with 200 ul Binding Buffer A and 20 ul MAP Solution A see Starting a Run page 19 15 InviMag Tissue DNA Mini Kit KF96 0515 Protocol 3 Isolation of genomic DNA from formalin fixed tissue or formalin fixed paraffin embedded tissue FFPE Please read the instructions carefully and conduct the prepared procedure Attention Please be aware that the Binding Buffer A has to be prepared see instructions page 12 Note DNA is degraded by treatment with formalin The DNA isolated from formalin fixed tissues will be of lower quality than the DNA isolated from deep frozen tissue samples Tissues stored in buffered formalin pH 7 are more suitable than samples from non buffered formalin The DNA is more damaged in non buffered samples Note that DNA isolated from formalin fixed tissue is usually of lower molecular weight than DNA from fresh or frozen samples The degree of fragmentation depends on the type and age of the sample and the conditions used for fixation If the tissue sample is treated with formalin and subsequently embedded in paraffin please follow protocol 2 see page 15 for deparaftination of the sample Afterwards the resulting formalin fixed starting material should be treated as described in the protocol given here 1 Starting Mate
37. ts and equipment to be supplied by user o Measuring cylinder 250 ml o Vortexer o Pipette and pipette tips o ddH O o Disposable gloves o 96 100 ethanol o Reaction tubes 1 5 or 2 0 ml o Isopropanol The InviMag Tissue DNA Mini Kit KF96 is validated with 2 Propanol Rotipuran gt 99 7 p a ACS ISO Order no 6752 from Carl Roth Possible suppliers for Ilsopropanol Carl Roth Applichem Sigma Aldrich 2 Propanol 2 Propanol fur die Molekularbiologie 2 Propanol Rotipuran gt 99 7 p a ACS ISO Order No A3928 Order No 59304 1L F Order No 6752 42 InviMag Tissue DNA Mini Kit KF96 0515 Important indications for the InviMag Tissue DNA Mini Kit KF96 1 From liver samples only use up to 20 mg of tissue Excessive amount of tissue will result in inefficient lysis 20 mg tissue will provide an average yield of 4 35 ug genomic DNA Incomplete removal of the cell culture media will dilute the lysate and affect lysis Optimal disruption of the tissue sample is important for obtaining maximum yields and purity of the genomic DNA The most common method of cell disruption is by grinding with a mortar and pestle using liquid nitrogen With this method it is possible to reduce the incubation time with Proteinase K Alternatively a homogenizer for disruption of freshly extracted tissue samples can be used Homogenization of the tissue sample in Lysis Buffer G is recommended The simultaneous isolation of RNA by the InviMag
38. ul Proteinase K Important Vortex the tube for 5 sec An incomplete mixing will reduce quality and yield of the isolated DNA 2 Incubate the sample at 56 C for 2h while continuously shaking e g by using a thermomixer 3 During lysis prefill all plates with required buffers and appropriate volumes see Starting a Run page 19 4 After lysis centrifuge the sample for 2 min at max speed and transfer the cleared supernatant carefully into the cavity of the Binding Plate prefilled with 200 ul Binding Buffer A and 20 ul MAP Solution A see Starting a Run page 19 B Sample lysis in a Deep Well Plate not provided 1 Transfer the starting material after deparaffination See above into the cavities of the Deep Well Plate and add 400 ul Lysis Buffer G and 25 ul Proteinase K 2 Seal the Deep Well Plate with a Sealing Foil Important Note Vortex the Deep Well Plate 10 times An incomplete mixing will reduce quality and yield of the isolated DNA 3 Incubate the Deep Well Plate at 56 C for 2h use e g hybridization oven or incubator until lysis is complete Important Note Vortex the Deep Well Plate each 20 minutes for 20 sec Vortexing is essentially for an efficient lysis step No vortexing will lead to a dramatic reduction of the final yield 5 During lysis prefill all plates with the required buffers and appropriate volumes see Starting a Run page 19 6 After lysis centrifuge the Deep Well Plate wi
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