InviMag Blood DNA Mini Kit/ KF96 User manual
Contents
1. 90 ml 360 ml 30 ml 150 ml magnets wo Initial steps Catalogue No Lysis Buffer HLT Binding Solution Add 60 ml of 99 7 isopropanol to each bottle Wash Buffer HLT and mix thoroughly Add 105ml of 96 100 ethanol to the bottle Wash Buffer Il and mix thoroughly Add 90 ml of 96 100 ethanol to the bottle Wash Buffer M and mix thoroughly Add 1 1 ml of distilled water to each Proteinase K tube mix thoroughly until completely dissolving and store at 20 C Fill 30 ml Isopropanol molecular biologic grade into the empty bottle Add 240 ml of 99 7 isopropanol to each bottle Wash Buffer HLT and mix thoroughly Add 420 ml of 96 100 ethanol to the bottle Wash Buffer Il and mix thoroughly Add 450 ml of 96 100 ethanol to the bottle Wash Buffer M and mix thoroughly Add 10 5ml of distilled water to each Proteinase K tube mix thoroughly until completely dissolving and store at 20 C Fill 120 ml Isopropanol molecular biologic grade into the empty bottle Elution Plates and Tip Plates are identical Use one provided Elution Plate as a Tip Plate InviMag Blood DNA Mini Kit KF96 0515 Kit contents of InviMag Blood DNA Mini Kit KF96 w o plastic store the MAP Solution B 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 Catalogue No 7431300150 7431300250 are elan empty bottle empty bottle Heda final2. Stratecee molecular User manual InviM g Blood DNA Mini Kit KF96 for use on KingFisher 96 and KingFisher Flex Thermo Fisher for automated purification of total DNA from up to 200 ul of whole blood samples buffy coat non mammalian blood bone marrow and swabs with magnetic beads 7431300X00 aaaf STRATEC Molecular GmbH D 13125 Berlin Instruction for InviMag Blood DNA Mini Kit KF96 The InviMag Blood DNA Mini Kit KF96 combines the advantages of the innovative InviMag technology with easy handling of magnetic particles in combination with KingFisher instruments for an efficient and reliable isolation of genomic DNA from blood in a high purity The InviMag Blood DNA Mini Kit KF96 is the ideal tool for a semi automated isolation and purification of DNA genomic and mitochondrial from 200 ul whole blood samples stabilized with EDTA citrate but not heparin buffy coat non mammalian blood cerebrospinal fluid CSF bone marrow and swabs in a 96 well format The kit is designed for use on KF96 and or KFflex96 workstations from Thermo Scientific The interplay of the DNA extraction and purification chemistry provided by the InviMag Blood DNA Mini Kit KF96 was intensely tested and validated The DNA binding magnetic particles are characterized by a high surface area uniform size distribution good suspension stability and therefore are highly suitable for high throughput processing Due to the h
3. The chemicals and the plastic parts are for laboratory use only they must be stored in the laboratory and must not be used for purposes other than intended The product with its contents is unfit for consumption InviMag Blood 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 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 product 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 Blood 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 be considered infectious and be handled and discarded according to local safety regulations European Community risk and safety phrases for the components of the InviMag Blood DNA Mini Kit KF96 to which they apply are listed below as follows Wash Buffer I contains guanidine thiocyanate which is an irritant
4. MAP Solution B by vigorously vortexing 5 Choose either the KF96 protocol InviMag Blood KF96 or the KFflex96 protocol InviMag Blood KFflex96 depending on the used instrument and press the START button 6 Insert all prefilled plates into the instrument by following the specifications printed on the display and confirm every plate loading step by pressing the START button 7 After all prefilled plates have been loaded press the START button to initialize the assay file The run will take approximately 54 min Important After lysis a pause step occurs and 230 ul Binding Solution and 40 ul MAP Solution B have to be added to each sample containing cavity of the Binding Plate To achieve that the plate is transported to the initial loading position After adding both reagents reinsert the plate into the instrument and confirm this step by pressing the START button again The instrument will continue with the extraction without any further user interaction Watch out that the orientation of the reinserted plate is correct 16 InviMag Blood DNA Mini Kit KF96 0515 The following extraction steps run automatically on the KingFisher instrument Lysis of the blood cells Automatically sample mixing for 15 min at elevated temperature Adjustment of Binding condition Magnetic Beads MAP Solution B and Binding Solution are added to the lysed sample mixture Binding of the DNA Automatically sample mixing
5. Name Elution Buffer M Dispensed reagents Lysis Plate Name Isopropanol MAP Solution B Well volume pl Well volume pl 200 200 50 Well volume ul 800 Well volume ul 00 Well volume ul 900 Well volume ul 100 Step Adjust Binding Condition Adjust Binding Condition 18 For self programming the KF96 KFflex96 instrument KingFisher 96 KF 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 pl Microtiter DW 96 plate Total reagent volume ul King Fisher 96 KF plate Total reagent volume ul Microtiter DW 96 plate Well volume pl F 230 F 40 InviMag Blood DNA Mini Kit KF96 0515 Type Type Sample Reagent Reagent Type Reagent Type Reagent Type Reagent Type Reagent Total reagent volume ul Steps data mt Tipl Pick Up Lysis Step Beginning of step Mixing heating End of step Adjust Binding Condition Reagent s Binding Step Beginning of step Mixing heating End of step Washing Step 1 Beginning of step Mixing heating End of step Washing Step 2 Beginning of step Mixing heating End of step 96 DW tip comb Tip Plate Lysis Plate Precollect Release beads Mixing time speed Heating temperature C Preheat Postmix Collect beads Lysis Plate Message D
6. for 5 min MAP Solution B separation Moving of the MAP Solution B into the Washing Plate 1 First Washing Automatically sample mixing for 3 min MAP Solution B separation Moving of the MAP Solution B into the Washing Plate 2 Second Washing Automatically sample mixing for 2 min MAP Solution B separation Moving of the MAP Solution B into the Washing Plate 3 Third Washing and Drying Automatically sample mixing for 90 s MAP Solution B separation Drying the MAP Solution B outsight the plate for 5 min Moving of the MAP Solution B into the Elution Plate Elution of the DNA Incubation of the MAP Solution B into the Elution Plate for 10 min by mixing at elevated temperatures MAP Solution B separation The MAP Solution B will then be automatically removed into the wells of Washing Plate 3 disposal Important Note After finishing the extraction protocol the Elution Plate contains the extracted DNA We recommend store the DNA at 20 C If the extracted DNA contains carryover of magnetic particles transfer the DNA to a 1 5 ml reaction tube centrifuge at maximum speed 13000 rom for 1 min and transfer the DNA containing supernatant into a new tube 17 InviMag Blood DNA Mini Kit KF96 0515 Reagent info Tip Plate Name Lysis Plate Name Sample Lysis Buffer HLT Proteinase K Wash Plate 1 Name Wash Buffer HLT Wash Plate 2 Name Wash Buffer M Wash Plate 3 Name Wash Buffer II Elution Plate
7. h samples may be stored at 4 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 If cryoprecipitates formed during thawing of frozen samples are visible avoid aspirating them Various different primary tubes blood collection system e g Sarstedt Greiner and anticoagulants except heparin can be used to collect blood samples for the InviMag Blood DNA Mini Kit KF96 procedure Buffy coat is a whole blood fraction of enriched leukocyte cells To prepare and extract a buffy coat layer the following procedure is recommended The use of a whole blood sample anticoagulants EDTA citrate not heparin with a sedimented cellular fraction from staying overnight at 4 C is recommended The resulting bright mid section overlaid by the clear plasma is buffy coat containing concentrated leukocytes that can be easily distinguished from the erythrocytes in the bottom layer An enrichment factor of 10 is expected from such a procedure Due to the enriched leukocyte content be aware to avoid overloading the system CSF cerebrospinal fluid and bone marrow Best results are obtained with fresh material that can be stored for 2 3 h at 4 C for short term storage For long term storage freezing at 20 C is recommended Dried samples have to be stored at 4 C in a dry surrounding Swabs The protocol works with fresh prepared swabs as well as with dri
8. 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 genomic DNA from cultured or isolated cells from tissue blood cards dried blood stains urine nor from stool sample bacteria fungi parasites or the purification of total RNA The application of the kit for isolation and purification of viral DNA has not been evaluated The included chemicals are only useable once Differing of starting material or flow trace may lead to inoperability therefore neither a warranty nor guarantee in this case will be given neither implied nor express 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 detection thereof
9. marrow swabs up to 200 ul rinsed liquid from swab The semi automated DNA isolation process is based on the interaction of 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 The procedure requires only minimal interaction by the user thus allowing safe handling of potentially infectious samples Sample cross contamination and reagent cross over is effectively eliminated by the automated purification process The KingFisher instrument uses magnetic rods to transport the DNA binding magnetic particles through the various purification phases such as binding washing drying and elution The volume of buffers and other liquids required for isolation is reduced to a minimum Eliminating most of the direct liquid handling and increasing the automation level results in a fast reliable and robust technique After a sample specific cell lysis on the instrument using the Lysis Buffer HLT and Proteinase K optimal binding conditions are adjusted by addition of Binding Solution The released DNA binds to the simultaneously added MAP Solution B magnetic particles and is separated from solution by magnetic rods controlled by the KingFisher machine Subsequent to three washing steps using Wash Buffer HLT Wash Buffer M and Wash Buffer II the DNA is finally eluted in Elution Buffer M Due to the high pur
10. swab inside the wall of the transportation tube and discard it 2 Transfer 200 ul from the transportation media jetting liquid into a 1 5 ml reaction tube not provided If the sample volume is lower than 200 ul adjust with 1x PBS or distilled water 3 Add 200 ul Lysis Buffer HLT and 20 ul Proteinase K to each sample containing cavity of the Lysis Plate 4 Proceed with prefilling of the plates and preparation of the instrument see Starting a Run page 16 15 InviMag Blood DNA Mini Kit KF96 0515 Starting a run on a KF96 KFflex96 instrument Important For working with the KingFisher instruments please carefully read the manufacturer s instructions before use 1 Turn on the KF96 or KFflex96 instrument Note Use one provided Elution Plate as Tip Plate These plates are identical 2 Prefill all Deep Well Plates with the appropriate buffers and volumes as indicated below 3 Tip Plate 4 Lysis Plate Washing plate 1 Washing plate 2 Washing plate 3 Elution Plate Place the provided Tip Comb for DW magnets on a 200 ul Elution Plate these are identical Add 200 ul sample 20 ul Proteinase K and 200 ul Lysis Buffer HLT After lysis a pause step occurs and 230 ul Binding Solution and 40 ul MAP Solution B have to be added to each sample containing cavity Add 800 ul Wash Buffer HLT Add 900 ul Wash Buffer M Add 900 ul Wash Buffer Il Add 100 ul Elution Buffer M Important Mix the bottle with the
11. Lysis Buffer HLT Wash Buffer HLT Proteinase K contains guanidine hydrochloride H302 315 319 P280 305 351 338 H315 319 334 335 P280 305 351 338 310 405 H315 Causes skin irritation H319 Causes serious eye irritation 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 Remove contact lenses and continue rinsing P310 Immediately calla POISON CENTER or doctor physician P405 Store locked up 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 InviMag Blood DNA Mini Kit KF96 0515 Product characteristics of the InviMag Blood DNA Mini Kit KF96 The InviMag Blood DNA Mini Kit KF96 procedure is the ideal tool for an efficient DNA extraction and purification from fresh or frozen whole blood samples non mammalian blood buffy coat CST bone marrow and swabs in a convenient 96 well format using magnetic beads and the KF96 KFflex96 instrument 1 200 ul fresh or frozen human or other mammalian whole blood 3 8 ug depends on the blood sample kind storage and source 1 200 ul cerebrospinal fluid 1 30 ul buffy coat 1 25 ul fresh frozen or old non mammalian blood 1 20 ul bone
12. RATEC 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 Blood DNA Mini Kit KF96 have been 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 Blood 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 InviMag Blood 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 Blood DNA Mini Kit KF96 is designed for semi automated extraction and ourification of total genomic and mitochondrial DNA from 8 96 whole blood or blood related samples using magnetic beads and the KF96 or KFlex96 instrument The nucleic acid isolation protocol is suitable for routinely walk away automated preparation of DNA from fresh or frozen whole blood samples buffy coats non mammalian bloods cerebrospinal fluids CSF bone marrow and swabs For reproducible and high yi
13. at 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 its function in various downstream applications Damaged DNA performs poorly in applications such as 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 80 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 20 C may cause shearing particularly if the DNA is exposed to repeated freezing and thawing 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 air dry DNA 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 incubate the DNA in buffer overnight at 2 8 C Minimize vortexing of genomic DNA because this can cause sheari
14. c acids Magnetic Separation Pure nucleic acids Elution Plates and Tip Plates are identically Use one provided Elution Plate as a Tip Plate 13 InviMag Blood DNA Mini Kit KF96 0515 Lysis Procedures Protocol 1 Isolation of genomic DNA from up to 200 ul of whole blood up to 30 ul of buffy coat Please read the instructions carefully and conduct the prepared procedure Important Note Samples with a smaller volume than 200 ul must be adjusted to a final volume of 200 ul using either 1x PBS or distilled water 1 Transfer 200 ul of whole blood or 30 ul buffy coat into a free cavity of the Lysis Plate and add 200 ul Lysis Buffer HLT and 20 ul Proteinase K to each sample containing cavities 2 Proceed with prefilling of the plates and preparation of the instrument see Starting a Run page 16 Protocol 2 Isolation of genomic DNA from up to 30 ul of non mammalian blood Please read the instructions carefully and conduct the prepared procedure Important note Bird e g chicken or fish blood contains nucleated erythrocytes Therefore only 10 15 ul of starting material should be used for isolation 1 Transfer max 30 ul of non mammalian blood not heparin stabilized into the cavities of the Lysis Plate Adjust the sample volume to 200 ul with 1x PBS or distilled water 2 Add 200 ul Lysis Buffer HLT and 20 ul Proteinase K to sample containing cavities of the Lysis Plate 3 Proceed with prefilling of the plates and
15. ed swabs Please note that dried swab samples are often characterized by isolation of apoptotic DNA visible on agarose gel as a typical apoptotic DNA band pattern The protocol has not been validated for isolation of DNA from swabs which are stored in special storage buffers from other providers 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 Principle and procedure The InviMag Blood DNA Mini Kit KF96 procedure comprises following steps o lysis of blood cells and protein digestion o binding the genomic DNA to magnetic beads o washing of the bead bound DNA and elimination of ethanol o elution of genomic DNA After lysis the DNA binds to the magnetic beads whereas contaminations and enzyme inhibitors are efficiently removed during the following three washing steps and highly purified DNA is eluted in Elution Buffer D This manual contains 4 protocols see page 14 15 Lysis Samples with a volume lower than 200 ul should be adjusted to 200 ul using 1x PBS or distilled water before starting the protocol For optimal results samples must be equilibrated at room temperature before lysis InviMag Blood DNA Mini Kit KF96 0515 Samples are lysed at elevated temperatures in the presence of Lysis Buffer HLT and Proteinase K In case of large number of samples the preparation of a mast
16. elds appropriate sample storage is essential see Sampling and storage of the starting material page 10 Common blood collection tubes not provided and anticoagulants EDTA citrate but not heparin can be used to assemble a set of blood samples All utilities reagents and plastic ware necessary for preparation of total DNA are provided by the InviMag Blood DNA Mini Kit KF96 in different package sizes The procedure of the InviMag Blood DNA Mini Kit KF96 is optimized for the isolation of total DNA from up to 200 ul starting material For samples of a smaller volume than 200 ul please adjust to a total sample volume of 200 ul with 1x PBS prior to the start of an isolation protocol THE PRODUCT IS INDENTED FOR USE BY PROFESSIONAL USERS ONLY 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 results 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
17. er mixture with a volume 5 greater than that required for the processing of all samples is recommended Binding of the genomic DNA After adding Binding Solution and MAP Solution B the DNA is bound to the surface of the magnetic beads Removing residual contaminants Contaminants are efficiently removing using Wash Buffer HLT Wash Buffer M and Wash Buffer II while the DNA remains bound to the magnetic beads Elution The DNA is eluted in Elution Buffer M and is ready to use in different subsequent downstream applications like o PCR amplification o digestion with restriction enzymes o Southern hybridizations o HLA typing etc Yield and Quality The amount of purified DNA in the InviMag Blood DNA Mini Kit KF96 procedure from whole blood depends on the leucocytes content the sample source transport storage and age Typically a 200 ul sample of whole blood cells samples with elevated white blood cell WBC counts ranging from 3x10 to 1x10 cells ml from a healthy individual will yield 3 8 ug of DNA If the whole blood sample is mixed with anticoagulant containing buffer solutions the overall leukocyte concentration decreases and the yield of the DNA extraction procedure is reduced 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 Important notes Important points before sta
18. ftware 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 develooment 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 mcg epee 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 available 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 29 InviMag Blood DNA Mini Kit KF96 0515 General notes on handling DNA Nature of DNA The length and delicate physical nature of DNA requires careful handling to avoid damage due to shearing and or enzymatic degradation Other conditions th
19. g material is stored at appropriate conditions 20 C 80 C avoid multiple thawing and freezing cycles of the material Due to the very gentle isolation procedure it may occur that isolated genomic DNA forms a clew To overcome this the first primary PCR denaturation step at 95 C should be prolonged to 5 min Increase drying time for removal of ethanol in the assay file Check 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 equilibrated at room temperature before usage Centrifuge at full soeed for 1 min and transfer supernatant to a new tube InviMag Blood DNA Mini Kit KF96 0515 Appendix KingFisher Bindlt Software 3 2 or higher versions Bindlt software 3 2 or 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 so
20. ge Invisorb DNA Blood Mini HTS 96 Kit C 4 x 96 preparations 1031300300 Invisorb DNA Blood Mini HTS 96 Kit C 24 x 96 preparations 1031300400 using the X tractor Gene Corbett Robotics Invisorb Blood Mini HTS 96 Kit X 4 x 96 preparations 7131310300 Invisorb Blood Mini HTS 96 Kit X 24 x 96 preparations 7131310400 Possible suppliers for lsopropanol Carl Roth Applichem Sigma 2 Propanol 2 Propanol fur die Molekularbiologie 2 Propanol Rotipuran gt 99 7 p a ACS ISO Order no A3928 Order no 6752 24 Order no 59304 1L F InviMag Blood 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 1G3a02 05 2015
21. igh purity the isolated DNA is ready to use for in vitro diagnostic analysis in a broad panel of downstream applications or can alternatively be stored at 20 C for subsequent use The kit is neither validated for the isolation of genomic DNA from cultured or isolated cells from tissues blood cards dried blood stains urine nor from stool samples bacteria fungi parasites total RNA The application of the kit for isolation and purification of viral DNA has not been evaluated C 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 Blood DNA Mini Kit KF96 0515 Table of contents Kit contents of InviMag Blood DNA Mini Kit KF96 3 Kit contents of InviMag Blood DNA M
22. ini Kit KF96 w o plastic 4 Symbols 5 Storage 5 Quality control 5 Intended use 6 Product use limitation 6 Safety information 7 Product characteristics of the InviMag Blood DNA Mini Kit KF96 8 Product validation 9 Sampling and storage of starting material 10 Principle and procedure 10 Yield and Quality 11 Important notes 11 Preparing reagents and buffers 12 Reagents and equipment to be supplied by user 12 Scheme of the InviMag Blood DNA Mini Kit KF96 13 Lysis Procedures 14 Protocol 1 Isolation of genomic DNA from up to 200 ul of whole blood up to 30 ul of buffy coat 14 Protocol 2 Isolation of genomic DNA from up to 30 ul of non mammalian blood 14 Protocol 3 Isolation of genomic DNA from CSF and bone marrow 14 Protocol 4 Isolation of genomic DNA from swabs or rinsed liquid from swabs 15 Starting a run on a KF96 KFflex96 instrument 16 For self programming the KF96 KFflex96 instrument 18 Troubleshooting 21 Appendix 22 General notes on handling DNA 23 Ordering information 24 InviMag Blood DNA Mini Kit KF96 0515 Kit contents of InviMag Blood DNA Mini Kit KF96 store the MAP Solution B at 2 8 C Store dissolved Proteinase K at 20 C Store all other kit components at room temperature RT 1x 96 extractions 5x 96 extractions 7431300100 7431300200 fill with 99 7 empty bottle empty bottle UA final volume 30 ml final volume 120 ml Proteinase K EENS nn working solution MAP Solution B 4x 1 1 ml 2x 10 5 ml
23. ispensing volume ul Name Volume ul Name Volume ul Lysis Plate Precollect Release time speed Mixing time speed Heating during mixing Postmix Collect count Collect time s Wash Plate 1 Precollect Release time speed Mixing time speed Heating during mixing Postmix Collect count Collect time s Wash Plate 2 Precollect Release time speed Mixing time speed Heating during mixing Postmix Collect count Collect time s 19 No Yes 00 15 00 Medium 75 Yes No No Add Isopropanol MAP Sol B 270 Isopropanol 230 MAP Solution B 40 No 00 00 10 Fast 00 05 00 Medium nz Z oo No 00 00 10 Fast 00 03 00 Fast No o MM nn vt A Z No 00 00 10 Fast 00 02 00 Fast No o ke EN Ui AN InviMag Blood DNA Mini Kit KF96 0515 Washing Step 3 Beginning of step Mixing heating End of step Drying Step Elution Step Beginning of step Mixing heating End of step Bead Removal Step Leave Wash Plate 3 Precollect Release time speed Mixing time speed Heating during mixing Postmix Collect count Collect time s Wash 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 Wash Plate 3 Release time speed Tip Plate 20 No 00 00 10 Fast 00 01 30 Fast uz Z O Oo 00 05 00 Outside well
24. ity the eluted total genomic and mitochondrial DNA is ready to use in a broad panel of downstream applications PCR Real time PCR PCR qPCR Restriction Enzyme Digestion HLA Typing Southern Blot The InviMag Blood DNA Mini Kit KF96 is supplied with a comprehensive manual describing four protocols page 14 15 for DNA purification from different sample sources For the semi automated isolation of genomic DNA from 200 ul blood using magnetic particles in a single well format for up to 15 samples per run STRATEC Molecular offers the InviMag Blood DNA Mini Kit KFmL for use on a KingFisher mL instrument and for 1 ml whole blood the InviMag Blood DNA Midi Kit KFflex24 O O O 0 The PCR process is covered by US Patents 4 683 195 and 4 683 202 and foreign equivalents owned by Hoffmann La Roche AG InviMag Blood DNA Mini Kit KF96 0515 For the isolation of DNA from a single blood sample STRATEC Molecular offers the Invisorb Spin Blood Mini Kit or for 8 96 samples the Invisorb DNA Blood Mini HTS 96 Kits for use on a centrifuge or robotic station see Ordering information page 24 For further information please contact 49 0 30 9489 2901 or 2910 in Germany and 49 0 30 9489 2907 from foreign countries or ask your local distributor Product validation PCR inhibitor and cross contamination test To maximize the detection of any potential contamination event positive and no template controls NT Cs were ar
25. lt operating instructions Temperature limitation OS ERIE Do not reuse Storage All buffers and kit contents of the InviMag Blood DNA Mini Kit KF96 except dissolved Proteinase K and MAP Solution B should be stored at room temperature and are stable for at least 12 months at these conditions Proteinase K Dissolved Proteinase K must be stored at 20 C Dividing the Proteinase K into aliquots and storage at 20 C is recommended Avoid multiple freezing and thawing cycles because this will lead to decreased enzymatic activity MAP Solution B The magnetic particles should be stored at 2 8 C Wash Buffer HLT Wash Buffer M Wash Buffer Il Binding Solution Wash Buffers and Binding Solution charged with either ethanol or isopropanol should be stored well sealed at room temperature If any precipitates are visible within the provided solutions solve them by carefully warming up to 30 C Room temperature RT is defined as range from 15 30 C Quality control and product warranty STRATEC Molecular warrants the correct function of the InviMag Blood DNA Mini Kit KF96 for applications as described in this manual Purchaser must determine the suitability of the product for its particular use Should any product fail to perform the applications as described in the manual STRATEC Molecular will check the lot and if STRATEC Molecular investigates a problem in the lot STRATEC Molecular will replace the product free of charge ST
26. ng Avoid vigorous pipetting Pipetting genomic DNA through small tip openings may cause shearing or nicking One way to decrease shearing of genomic DNA is to use special tips that have wide openings especially designed for pipetting genomic DNA Regular pipette tips pose no problem for plasmid or other small DNA 53 InviMag Blood DNA Mini Kit KF96 0515 Ordering information Product Package size InviMag Blood DNA Mini Kit KF96 1 x 96 preparations InviMag Blood DNA Mini Kit KF96 5 X 96 preparations KingFisher 96 and consumables KingFisher 96 Magnetic Particle Processor 100 240V 50 60Hz including one magnetic head KingFisher 96 Head for Deep Well plate KingFisher 96 tip comb for PCR magnets 8 x 10 pcs box KingFisher 96 tip comb for KF magnets 10 x 10 pcs box KingFisher 96 tip comb for DW magnets 10 x 10 pcs box KingFisher 96 KF plate 200ul 48 plates box Microtiter deep well 96 plate 50 plates box Catalogue No 7431300100 7431300200 5400500 24073430 97002514 97002524 97002534 97002540 95040450 Related products Package size Catalogue No InviMag Blood DNA Mini Kit KFmL 15 preparations 2431110100 InviMag Blood DNA Mini Kit KFmL 75 preparations 2431110200 Invisorb Spin Blood Mini Kit 50 preparations 1031100200 Invisorb Spin Blood Mini Kit 250 preparations 1031100300 Invisorb Spin Blood Midi Kit 00 preparations 1031130200 Invisorb Blood Universal Kit 900 ml 1031150200 using a centrifu
27. olve any precipitates by incubation at 30 C Swirl gently to avoid foaming Lysis Buffer HLT and Elution Buffer M are ready to use Prepare all other reagents as indicated below 1 x 96 DNA extractions Add 60 ml of 99 7 isopropanol to each bottle Wash Buffer HLT and mix thoroughly Add 105 ml of 96 100 ethanol to the bottle Wash Buffer Il and mix thoroughly Add 90 ml of 96 100 ethanol to the bottle Wash Buffer M and mix thoroughly Add 1 1 ml of distilled water to each Proteinase K tube mix thoroughly until completely dissolving and store at 20 C Fill 30 ml Isopropanol molecular biologic grade into the empty bottle 5 x 96 DNA extractions Add 240 ml of 99 7 isopropanol to each bottle Wash Buffer HLT and mix thoroughly Add 420 ml of 96 100 ethanol to the bottle Wash Buffer Il and mix thoroughly Add 450 ml of 96 100 ethanol to the bottle Wash Buffer M and mix thoroughly Add 10 5 ml of distilled water to each Proteinase K tube mix thoroughly until completely dissolving and store at 20 C Fill 120 ml Isopropanol molecular biologic grade into the empty bottle Reagents and equipment to be supplied by user Measuring cylinder 250 ml Pipette and pipette tips Disposable gloves Reaction tubes 1 5 ml or 2 0 ml dd H2O 96 100 ethanol 99 7 isopropanol O O O O O O The InviMag Blood DNA Mini Kit KF96 is validated with 2 Propanol Rotipuran gt 99 7 p a ACS ISO Order no 6752 from Carl Roth P
28. ossible suppliers for Isopropanol CATION Applichem Sigma 2 Propanol 2 Propanol f r die Molekularbiologie 2 Propanol Rotipuran gt 99 7 p a ACS ISO Order no A3928 Order no 59304 1L F Order no 6752 InviMag Blood DNA Mini Kit KF96 0515 Scheme of the InviMag Blood DNA Mini Kit KF96 Please read protocols prior the start of the preparation carefully Transfer 200 ul Lysis Buffer HLT and 200 ul sample into a cavity of a 2 ml Deep Well Plate refers as Lysis Plate and 20 ul Proteinase K Prefill all remaining plates with required buffers and appropriate volumes Tip Plate Lysis Plate Washing Plate 1 Washing Plate 2 Washing Plate 3 Elution Plate Insert the KF96 Tip Comb for DW magnets on a Tip Plate Add 200 ul Lysis Buffer HLT 200 ul sample 20 u Proteinase K Add 800 ul Wash Buffer HLT to a 2 0 ml Deep Well Plate Add 900 ul Wash Buffer M to a 2 0 ml Deep Well Plate Add 900 ul Wash Buffer Il to a 2 0 ml Deep Well Plate Add 100 ul Elution Buffer M to the Elution Plate same size as Tip Plate Please read the protocols carefully prior to the start of the preparation procedure The following steps are performed on the KingFisher instrument Lysis of the sample After lysis a pause step occurs and 230 ul Binding Solution and 40 ul MAP Solution B have to be added Nucleic acids bind to magnetic particles Washing of the particle fixed nucleic acids Magnetic separation Elution of nuclei
29. preparation of the instrument see Starting a Run page 16 Protocol 3 Isolation of genomic DNA from CSF and bone marrow Please read the instructions carefully and conduct the prepared procedure Preparation of the starting material Fresh material o 1 200 ul fresh cerebrospinal fluid o 1 20 ul bone marrow Dried material for example on hematological slides o Moisten the dried material with a drop of PBS o Add 180 ul PBS to a 1 5 ml reaction tube not provided and scrape the cytological material into the tube using the edge of a clean slide o Dissolve the resulting sludge by pipetting up and down several times 1 Transfer the starting material into free cavities of the Lysis Plate Adjust the sample volume to 200 ul with 1x PBS or distilled water 2 Add 200 ul Lysis Buffer HLT and 20 ul Proteinase K to sample containing cavities of the Lysis Plate 3 Proceed with prefilling of the plates and preparation of the instrument see Starting a Run page 16 14 InviMag Blood DNA Mini Kit KF96 0515 Protocol 4 Isolation of genomic DNA from swabs or rinsed liquid from swabs Please read the instructions carefully and conduct the prepared procedure Dried swabs If the swab is delivered without transportation media rinse the swab in a 1 5 ml reaction tube filled with 200 300 ul cooled water or 1x PBS Mix for several minutes by shaking and continue with step 1 see below Rinsed swabs 1 Squeeze out the
30. ranged in alternating wells in a chessboard pattern Fig 1 Out of those samples Fig 2 shows a real time PCR run of the extracted DNA PCRs were done with a GAPDH Primer set in an in house SyBr Green assay on a Corbett Rotor Gene 3000 12 3 4 5 6 7 8 9 10 11 12 X JOI JOI JOI JOI JOL JO BOO 0 0 000 a Or JOL JOL JOL JOL JO DO 0e ee a JOL JOL JOL JOL JO zor JOL JO JOL IOL JOL a JOL JO JO JOL JOL IG HOO 0 00 Le Fig 1 Chessboard Pattern utilized for the cross contamination analysis test Samples red and NTC white arranged in alternating wells Norm Fluoro dF AT 0 Threshold 70 75 80 Fig 2 Real time PCR results from samples red and no samples controls yellow arranged in Chessboard NTC black and PTC pink are also shown Reproducibility ee ee DNA was isolated from 200 ul transfusion blood The ratio A 260 280 is between 1 94 0 06 The yield is about 3 5 0 3 ug InviMag Blood DNA Mini Kit KF96 0515 Sampling and storage of 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 health of the donor sample age kind of sample transport and storage conditions Blood and buffy coat Best results are obtained using fresh blood samples Mammalian blood samples stabilized with EDTA or Citrate can be stored at room temperature for 2 3 hours for short time storage up to 24
31. rting a protocol Immediately upon receipt of the product inspect the product and its components as well as the package for any apparent damages correct quantities and quality If there are any unconformities you have to notify STRATEC Molecular in writing with immediate effect upon inspection thereof 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 since their use may lead to poor kit performance o Always change pipet tips between different liquid transfers To avoid cross contaminations 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 gloves inmediately Do not combine components from 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 This kit should only be used by trained personnel O O O O O InviMag Blood DNA Mini Kit KF96 0515 Preparing reagents and buffers Before starting a run equilibrate all reagents to room temperature Where necessary gently mix and rediss
32. tube No 00 00 10 Medium 00 10 00 Slow 65 Yes o 1 ATZ 00 00 30 Fast InviMag Blood DNA Mini Kit KF96 0515 Troubleshooting Probable cause Comments and suggestions Low amount of extracted DNA Low concentration of extracted DNA Degraded DNA DNA does not perform well in downstream applications e g real time PCR or PCR Eluted DNA is brownish colored Insufficient lysis Incomplete elution Inhomogeneous amount of beads Too much Elution Buffer Incorrect storage of starting material Incorrect Wash Buffers Incorrect storage of starting material Old material No PCR result for genomic DNA Ethanol carryover during elution Salt carry over during elution Small part of the magnetic particles are left in the elution 21 Increase lysis time but prevent too long lysis time because this will decreases the yield or reduce amount of starting material Increase the volume of Elution Buffer M Ensure that the Elution Buffer M is transferred to the right cavity Mix MAP Solution B vigorously before use Elute the DNA in a lower volume of Elution Buffer M Ensure that the storage of starting material was correct Avoid repeated thawing and freezing cycles of the sample material Ensure that the correct amount of ethanol isopropanol is added to the Wash Buffers and storage is correct Ensure that the storage of starting material was correct Ensure that the startin
33. volume 30 ml final volume 120 ml Proteinase K 2x 1 1 ml 10 5 ml working solution i MAP Solution B 4x 1 1 ml 2x 10 5 ml 90 ml 360 ml 30 ml 150 ml 45 ml 180 ml 1 5 ml Receiver Tubes 2x 50 pieces 10 x 50 pieces Add 60 ml of 99 7 Isopropanol to each bottle Add 240 ml of 99 7 Isopropanol to each Wash Buffer HLT and mix thoroughly bottle Wash Buffer HLT and mix thoroughly Add 105 ml of 96 100 ethanol to the bottle Add 420 ml of 96 100 ethanol to the bottle Wash Buffer Il and mix thoroughly Wash Buffer Il and mix thoroughly Add 90 ml of 96 100 ethanol to the bottle Add 450 ml of 96 100 ethanol to the bottle Initial steps Wash Buffer M and mix thoroughly Wash Buffer M and mix thoroughly Add 1 1 ml of distilled water to each Add 10 5ml of distilled water to each Proteinase K tube mix thoroughly until Proteinase K tube mix thoroughly until completely dissolving and store at 20 C completely dissolving and store at 20 C Fill 30 ml Isopropanol molecular biologic Fill 120 ml Isopropanol molecular biologic grade into the empty bottle grade into the empty bottle Plastic to be supplied by user see order information 2 0 ml Deep Well Plate 4 pieces 20 pieces KF 96 Tip Comb for Elution Plates and Tip Plates are identical Use one provided Elution Plate as a Tip Plate InviMag Blood DNA Mini Kit KF96 0515 Symbols paal Manufacturer LOT Lot number Catalogue number Expiry date Consu
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