InviMag Blood DNA Mini Kit/ KFDuo User manual
Contents
1. 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 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 EDTA and citrate but not heparin can be used to collect blood samples for the InviMag Blood DNA Mini Kit KFDuo w o plastic 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 red colored 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 fres2. Stratecee molecular User manual InviMag Blood DNA Mini Kit KFDuo for use on KingFisher Duo Thermo Fisher Scientific 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 2431130X00 esal STRATEC Molecular GmbH D 13125 Berlin pe Instruction for InviMag Blood DNA Mini Kit KFDuo w o plastic The InviMag Blood DNA Mini Kit KFDuo 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 KFDuo is the ideal tool for a semi automated isolation and purification of DNA genomic and mitochondrial from 200 ul of whole blood samples stabilized with EDTA or citrate but not heparin buffy coat non mammalian blood cerebrospinal fluid CSF bone marrow and swabs in a 12 well format The kit is designed for use with the KFDuo workstation from Thermo Scientific The interplay of the DNA extraction and purification chemistry provided by the InviMag Blood DNA Mini Kit KFDuo was intensely tested and validated The DNA binding magnetic particles are characterized by a high surface area uniform size distribution and good suspension stability Therefore they are highly suitable for high throughput processing
3. 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 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 the starting material or flow trace may lead to inoperability Therefore neither a warranty nor guarantee in this case will be given implied or expressed 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
4. 25 ul fresh frozen or old non mammalian blood 1 20 ul bone marrow swabs up to 200 ul rinsed liquid from swab 2 6 ug depends on the blood about Aze0 A280 sample kind storage and source 60 min 1 7 2 1 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 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 magnetic particles MAP Solution B and is separated from solution by magnetic rods controlled by the KingFisher mac
5. distilled water before starting the protocol For optimal results samples must be equilibrated at room temperature before lysis 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 master 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 removed 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 KFDuo w o plastic procedure from whole blood depends on the leucocytes content 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 3 x 10 to 1 x 10 cells ml from a healthy individual will yield 2 6 ug of DNA If the whole blood sample is mixed with anticoagulant containing buffer solutions the overall leukocyte concentration dec
6. o Measuring cylinder 250 ml o dd H2O0 o Pipette and pipette tips o 96 100 ethanol o Disposable gloves o 99 isopropanol o Reaction tubes 1 5 ml or 2 0 ml The InviMag Blood DNA Mini Kit KFDuo w o plastic is validated with 2 Propanol Rotipuran gt 99 7 p a ACS ISO Order no 6752 from Carl Roth Possible suppliers for lsopropanol Carl Roth Applichem Sigma 2 Propanol f r die Molekularbiologie 2 Propanol Sane Order no A3928 Order no 59304 1L F Rotipuran gt 99 7 p a ACS ISO Order no 6752 10 InviMag Blood DNA Mini Kit KFDuo w o plastic 0515 Scheme of the InviMag Blood DNA Mini Kit KFDuo w o plastic Please read protocols prior the start of the preparation carefully Transfer 200 ul Lysis Buffer HLT and 200 ul sample into a free cavity of row A of the Working Plate and 20 ul Proteinase K Prefill the Working Plate and Elution stripe with the required buffers and appropriate volumes Working Plate Tip Comb Insert the KF Duo 12 Tip Comb into row B of the Working Plate Lysis Add 200 ul Lysis Buffer HLT 200 ul sample 20 u Proteinase K to row A of the Working Plate Washing Plate_1 Add 900 ul Wash Buffer HLT to row C of the Working Plate Washing Plate_2 Add 900 ul Wash Buffer M to row D of the Working Plate Washing Plate_3 Add 900 ul Wash Buffer II to row E of the Working Plate Elution Stripe Add 100 ul Elution Buffer M to the Elution stripe Please read the protocols carefully prio
7. 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 swab inside the wall of the transportation tube and discard it 2 Transfer 200 ul from the transportation media jetting liquid into a free cavity of row A of the Working Plate 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 Working Plate 4 Proceed with prefilling of the remaining rows see Starting a Run page 14 13 InviMag Blood DNA Mini Kit KFDuo w o plastic 0515 Starting a run on the KFDuo instrument Important For working with the KingFisher instruments please carefully read the manufacturer s 1 2 instructions before use Turn on the KFDuo instrument using the power switch Prefill the Working Plate and Elution stripe with the appropriate buffers and volumes as indicated below Working Plate Tip Comb Place the provided KFDuo 12 Tip Comb in row B of the Working Plate Lysis Add 200 ul sample 20 ul Proteinase K and 200 ul Lysis Buffer HLT to row A of the Working Plate After lysis a pause step will occur and 230 ul Binding Solution and 40 ul MAP Solution B have to be added to each sample contain
8. 1031150200 using a centrifuge 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 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 59304 1L F Order no 6752 22 InviMag Blood DNA Mini Kit KFDuo w o plastic 0515 Stratecee molecular STRATEC Molecular GmbH Robert R ssle 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 1G3a09 05 2015
9. Due to the high purity the isolated DNA is ready to use for in vitro diagnostic analysis or can alternatively be stored at 20 C 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 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 4 InviMag Blood DNA Mini Kit KFDuo w o plastic 0515 Table of content Kit contents of InviMag Blood DNA Mini Kit KFDuo w o plastic 3 Symbols 4 Storage 4 Quality control and product warr
10. ally infectious material we recommend working under laminar air flow until the samples are lysed o This kit should only be used by trained personnel Preparing reagents and buffers Before starting a run equilibrate all reagents to room temperature Where necessary gently mix and redissolve 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 8 x 12 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 Add 30 ml 99 7 isopropanol molecular biological grade into the empty bottle 40 x 12 DNA extractions Add 240ml 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 II 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 Add 120 ml 99 7 isopropanol molecular biological grade into the empty bottle Reagents and equipment to be supplied by user
11. 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 shearing 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 21 InviMag Blood DNA Mini Kit KFDuo w o plastic 0515 Ordering information Product Package size Catalogue No InviMag Blood DNA Mini Kit KFDuo w o 8 x 12 preparations 2431130150 plastic InviMag Blood DNA Mini Kit KFDuo w o 40 x 12 preparations 2431130250 plastic KingFisher Duo and consumables KingFisher Duo 5400100 KingFisher Duo 12 tip comb 50 pieces 5012501000 KingFisher Duo elution strip 40 pieces 5012501100 DeepWell plate 2 ml KingFisher 50 pieces 5012401700 Related products Package size Catalogue No InviMag Blood DNA Mini Kit KFmL 15 preparations 2431110100 InviMag Blood DNA Kit KFmL 75 preparations 2431110200 InviMag Blood DNA Mini Kit KF96 1 x 96 preparations 7431300100 InviMag Blood DNA Mini Kit KF96 5 x 96 preparations 7431300200 Invisorb Spin Blood Mini Kit 50 preparations 1031100200 Invisorb Spin Blood Mini Kit 250 preparations 1031100300 Invisorb Spin Blood Midi Kit 50 preparations 1031130200 Invisorb Blood Universal Kit 500 ml
12. anol final volume 30 ml final volume 120 ml Proteinase K working solution AK FORM MAP Solution B 4x 1 1 ml 2x 10 5 ml 90 ml 360 ml Wash Batter HET final volume 150 ml final volume 600 ml 30 ml 150 ml Wash Buffet M final volume 120 ml final volume 600 ml 45 ml 180 ml Wash Buffer ll final volume 150 ml final volume 600 ml Elution Buffer M 15 ml 60 ml 1 5 ml Receiver Tubes 2 x 50 pieces 10 x 50 pieces Sealing Foils 2 pieces 10 pieces Manual Initial steps 1 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 99 7 Isopropanol molecular biological grade into the empty bottle 1 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 99 7 Isopropanol molecular biological grade into the empty bottle Plastic to be supplied by user
13. anty 4 Intended use 5 Product use limitation 5 Safety information 6 Product characteristics of the InviMag Blood DNA Mini Kit KFDuo 7 Sampling and storage of starting material 8 Principle and procedure 8 Yield and Quality 9 Important notes 9 Preparing reagents and buffers 10 Reagents and equipment to be supplied by user 10 Scheme of the InviMag Blood DNA Mini Kit KFDuo w o plastic 11 Lysis Procedures 12 Protocol 1 Isolation of genomic DNA from up to 200 ul of whole blood up to 30 ul of buffy coat 12 Protocol 2 Isolation of genomic DNA from up to 30 ul of non mammalian blood 12 Protocol 3 Isolation of genomic DNA from CSF and bone marrow 12 Protocol 4 Isolation of genomic DNA from swabs or rinse liquid from swabs 13 Starting a run on a KFDuo instrument 14 For self programming the KFDuo instrument 16 Troubleshooting 19 Appendix 20 General notes on handling DNA 21 Ordering information 22 2 InviMag Blood DNA Mini Kit KFDuo w o plastic 0515 Kit contents of InviMag Blood DNA Mini Kit KFDuo 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 Important see ordering information at page 22 The needed KFDuo plastic is not included in the kit 8 x 12 extractions 40 x 12 extractions Catalogue Number 2431130150 2431130250 Lysis Buffer HLT 30 ml 120 ml Binding Solution empty bottle empty bottle fill with 99 7 Isoprop
14. e ul Blood_DNA Precollect Release time speed Mixing time speed Heating during mixing Postmix Collect count Collect time s Post temperature Blood_DNA Precollect Release time speed Mixing time speed Heating during mixing Postmix Collect count Collect time s Post temperature Blood_DNA Precollect Release time speed Mixing time speed Heating during mixing Postmix Collect count Collect time s Post temperature 17 InviMag Blood DNA Mini Kit KFDuo w o plastic 0515 B Tip Comb A Lysis No Yes 00 15 00 Medium 75 No No No A Lysis Add Isopropanol MAPs 270 Isopropanol 230 MAP Solution B 40 A Lysis No 00 00 10 Fast 00 05 00 Medium No No F 5 No C Wash 1 No 00 00 10 Fast 00 03 00 Fast No No F 4 5 No D Wash 2 No 00 00 10 Fast 00 02 00 Fast No No r 4 5 No Washing Step 3 Beginning of step Mixing heating End of step Drying Step Elution Beginning of step Mixing heating End of step Bead Removal Step Leave Blood_DNA Precollect Release time speed Mixing time speed Heating during mixing Postmix Collect count Collect time s Post temperature Blood_DNA Dry time Tip position Elution Precollect Release time speed Mixing time speed Heating temperature C Postmix Collect count Collect time s Post temperature Blood_DNA Release ti
15. h 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 dried swabs Please note that dried swab samples are often characterized by isolation of apoptotic DNA visible on agarose gel as a typical apoptotic DNA pattern The protocol has not been validated for isolation of DNA from swabs stored in storage buffers from other providers STRATEC Molecular will be released of its responsibilities if other sample materials than described in the Intended Use chapter are processed or if the sample preparation protocols are changed or modified Principle and procedure The InviMag Blood DNA Mini Kit KFDuo w o plastic procedure comprises following steps 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 O 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 M This manual contains 4 protocols see page 12 13 8 InviMag Blood DNA Mini Kit KFDuo w o plastic 0515 Lysis Samples with a volume lower than 200 ul should be adjusted to 200 ul using 1x PBS or
16. herein Any problems incidents or defects shall be reported to STRATEC Molecular immediately upon detection thereof The chemicals and the plastics are for laboratory use only They must be stored in the laboratory and not be used for other purposes than intended The product with its content is not intended for consumption 5 InviMag Blood DNA Mini Kit KFDuo w o plastic 0515 Safety information When and while working with chemicals always wear a suitable lab coat disposable gloves and protective goggles Avoid direct skin contact with reagents 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 KFDuo w o plastic 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 must be handled and discarded accordingly to local safety regulations European Community risk and safety phrases for the components of the InviMag Blood DNA Mini Kit KFDuo w o plastic to which they a
17. hine Subsequent to three washing steps using Wash Buffer HLT Wash Buffer M and Wash Buffer Il the DNA is finally eluted in Elution Buffer M Due to the high purity the eluted total genomic and mitochondrial DNA is ready to use for various downstream applications PCR real time PCR Restriction Enzyme Digestion HLA Typing Southern Blot The InviMag Blood DNA Mini Kit KFDuo w o plastic is supplied with a comprehensive manual describing four protocols page 12 13 for DNA purification from different sample sources For the semi automated isolation of genomic DNA from 200 ul blood using magnetic particles for up to 15 samples per run STRATEC Molecular offers the InviMag Blood DNA Mini Kit KFmL for use on a KingFisher mL instrument O O O The PCR process is covered by US Patents 4 683 195 and 4 683 202 and foreign equivalents owned by Hoffmann La Roche AG 7 InviMag Blood DNA Mini Kit KFDuo w o plastic 0515 For the isolation of DNA from single blood samples STRATEC Molecular offers the Invisorb Spin Blood Mini Kit or for 8 96 samples the Invisorb DNA Blood Mini HTS 96 Kit for use in a centrifuge see Ordering information page 22 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
18. ing cavity Wash 1 Add 900 ul Wash Buffer HLT to row C of the Working Plate Wash 2 Add 900 ul Wash Buffer M to row D of the Working Plate Wash 3 Add 900 ul Wash Buffer Il to row E of the Working Plate Elution Stripe Elution Add 100 ul Elution Buffer M to the Elution Stripe Important Mix the bottle with the MAP Solution B by vigorously vortexing 3 4 Choose the KF Duo assay InviMag Blood DNA KF Duo and press the START button Insert the Working Plate and Elution stripe into the instrument by following the specifications printed on the display After loading press the START button to initialize the assay The run will take approximately 60 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 row A of the Working Plate 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 14 InviMag Blood DNA Mini Kit KFDuo w o plastic 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 Solut
19. ion are added to the lysed sample mixture Binding of the DNA Automatically sample mixing for 5 min MAP Solution B separation Moving of the MAP Solution B into the Wash 1 position First Washing Automatically sample mixing for 3 min MAP Solution B separation Moving of the MAP Solution B into the Wash 2 position Second Washing Automatically sample mixing for 2 min MAP Solution B separation Moving of the MAP Solution B into the Wash 3 position 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 stripe Elution of the DNA Incubation of the MAP Solution B into the Elution stripe 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 Wash 3 disposal Important Note After finishing the extraction protocol the Elution stripe will contain the extracted DNA We recommend storing the DNA at 20 C If the extracted DNA contains carry over of magnetic particles transfer the DNA to a 1 5 ml reaction tube centrifuge at maximum speed 13000 rpm for 1 min and transfer the DNA containing supernatant into a new tube 15 InviMag Blood DNA Mini Kit KFDuo w o plastic 0515 For self programming the KFDuo instrument Reagent info Name Well volume pl Total reagent volume ul Type Sample 700 A Sa
20. malian blood not heparin stabilized into free cavities of row A of the Working Plate Adjust the sample volume to 200 ul using either 1x PBS or distilled water 2 Add 200 ul Lysis Buffer HLT and 20 ul Proteinase K to sample containing cavities of row A of the Working Plate 3 Proceed with prefilling of remaining rows see Starting a Run page 14 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 a free cavity of row A of the Working 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 Working Plate 3 Proceed with prefilling of remaining rows see Starting a Run page 14 12 InviMag Blood DNA Mini Kit KFDuo w o plastic 0515 Protocol 4 Isolation of genomic DNA from swabs or rinsed liquid from swabs Please read the instructions carefully and conduct
21. me speed Blood_DNA 18 InviMag Blood DNA Mini Kit KFDuo w o plastic 0515 B Wash 3 No 00 00 10 Fast 00 01 30 Fast Wash 3 00 05 00 Outside well tube A Elution No 00 00 10 Medium 00 10 00 Slow 65 No 4 5 No E Wash 3 00 00 30 Fast B Tip Comb Troubleshooting Problem Probable cause Comments and suggestions Low amount of extracted DNA Insufficient lysis Incomplete elution Inhomogeneous amount of beads 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 max 130 ul Ensure that the Elution Buffer M is transferred to the right cavity Mix MAP Solution B vigorously before use Low concentration of extracted DNA Too much Elution Buffer Incorrect storage of starting material Incorrect Wash Buffers 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 Degraded DNA Incorrect storage of starting material Old material Ensure that the storage of starting material was correct Ensure that the starting material is stored at appropriate conditions 20 C 80 C avoid multiple tha
22. mple Lysis Buffer HLT 200 Reagent Proteinase K 20 Reagent Well volume ul Total reagent volume ul Name Well volume ul Total reagent volume ul Wash Buffer HLT 00 Reagent Name Well volume pl Total reagent volume ul Type Wash Buffer M 00 Reagent Name Well volume pl Total reagent volume ul Type Wash Buffer II 00 Reagent Name Well volume ul Total reagent volume ul Type Name Well volume ul Total reagent volume ul Type Name Well volume ul Total reagent volume ul Type Name Well volume ul Total reagent volume ul Type Elution Buffer M 100 Reagent Dispensed reagents Name Step Well volume pl Total reagent volume ul NEEN Adjust Binding 350 g POR Conditions 4 F MAP Solution B Adjust binding 4 Conditions 16 InviMag Blood DNA Mini Kit KFDuo w o plastic 0515 Steps data It Tip1 ma oe oe Pick Up Lysis Step Beginning of step Mixing heating End of step Adjust Binding Conditions 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 KingFisher Duo 12 tip comb Blood_DNA Blood_DNA Precollect Release beads Mixing time speed Heating temperature C Postmix Collect beads Post temperature Blood_DNA Message Dispensing volume ul Name Volume ul Name Volum
23. 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 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 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
24. ot possible to import assay files created with Bindlt 3 2 into older software versions Please ask your local Thermo Scientific distributor for a Bindlt 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 at least the Bindlt software 3 0 for assay development because this software version includes the correct adjustments for this plate It is highly recommended to use Thermo Microtiter Deep Well plates with KF96 KFflex96 KFDuo workstations to ensure the best purification result Minimum system requirements for Bindlt Software 3 2 PC requirements Supported operating systems 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 port available 1 for KF96 KFflex96 connection Pointing device Mouse or equivalent is required CD ROM drive 1 for software installation only Monitor color settings XVGA monitor with at least 1024x768 resolution and a 16 bit color environment If you do not have the correct Service Packs installed you can download them from the Microsoft web pages http www microsoft com 20 InviMag Blood DNA Mini Kit KFDuo w o plastic 0515 General notes on handling DNA Nature
25. ozen whole blood samples buffy coat non mammalian blood cerebrospinal fluid CSF bone marrow and swabs For reproducible and high yields an appropriate sample storage is essential see Sampling and storage of the starting material page 8 Common blood collection tubes not provided and anticoagulants EDTA citrate but not heparin can be used to gather 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 KFDuo w o plastic The procedure of the InviMag Blood DNA Mini Kit KFDuo is optimized for the isolation of 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
26. pply are listed below as follows Wash Buffer I contains guanidine thiocyanate which is an irritant 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 innaled 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 call a 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 6 InviMag Blood DNA Mini Kit KFDuo w o plastic 0515 Product characteristics of the InviMag Blood DNA Mini Kit KFDuo w o plastic The InviMag Blood DNA Mini Kit KFDuo w o plastic 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 12 well format using magnetic beads and the KFDuo instrument 1 200 ul fresh or frozen human or other mammalian whole blood 1 200 ul cerebrospinal fluid 1 30 ul buffy coat 1
27. r 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 nucleic acids Magnetic Separation Pure nucleic acids 11 InviMag Blood DNA Mini Kit KFDuo w o plastic 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 row A of the Working Plate and add 200 ul Lysis Buffer HLT and 20 ul Proteinase K to each sample containing cavities 2 Proceed with prefilling of remaining rows see Starting a Run page 14 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 mam
28. reases 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 the manufacturers specifications Important notes Important points before starting a protocol Immediately upon receipt of the Product inspect the product and its components as well as the package for any apparent damages and correct quantities If there are any unconformities 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 6 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 9 InviMag Blood DNA Mini Kit KFDuo w o plastic 0515 o All centrifugation steps are carried out at room temperature o When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles o Discard contaminated gloves immediately o Do not combine components from different kits unless the lot numbers are identical o Avoid microbial contamination of the kit reagents o To minimize the risk of infections from potenti
29. see order information 2 0 ml KF Deep Well Plate i Bid KF Duo 12 Tip Comb 8 40 KF Duo Elution stripe 8 40 3 InviMag Blood DNA Mini Kit KFDuo w o plastic 0515 Symbols Lot Lot number Catalogue number Expiry date Consult operating instructions Temperature limitation D BRIE Do not reuse Storage All buffers and kit contents of the InviMag Blood DNA Mini Kit KFDuo w o plastic 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 KFDuo w o plastic for applications as described in this manual Purchaser must determine the sui
30. tability of the product for its particular use Should any product fail to perform in applications as described in the manual STRATEC Molecular will check the lot If a problem is detected 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 Blood DNA Mini Kit KFDuo w o plastic have been tested separately against predetermined specifications routinely on lot to lot to ensure consistent product quality In case of questions or problems regarding any aspect of InviMag Blood DNA Mini Kit KFDuo or other STRATEC Molecular products please do not hesitate to contact us For technical support or further information please contact from Germany 49 0 30 9489 2901 2910 from abroad 49 0 30 9489 2903 2907 or contact your local distributor 4 InviMag Blood DNA Mini Kit KFDuo w o plastic 0515 Intended use The InviMag Blood DNA Mini Kit KFDuo w o plastic is designed for semi automated extraction and purification of total genomic and mitochondrial DNA from 1 12 whole blood or blood related samples using magnetic beads and the KFDuo instrument The nucleic acid isolation protocol is suitable for walk away automated preparation of DNA from fresh or fr
31. wing and freezing cycles of the material DNA does not perform well in downstream applications e g real time PCR or PCR No PCR result for genomic DNA Ethanol carryover during elution Salt carry over during elution Due to the very gentle isolation procedure it may occur that isolated genomic DNA forms a clue 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 Eluted DNA is brownish colored Small part of the magnetic particles are left in the elution Centrifuge at full speed for 1 min and transfer supernatant to a new tube 19 InviMag Blood DNA Mini Kit KFDuo w o plastic 0515 Appendix KingFisher Bindlt Software 3 2 The KingFisher Bindlt Software 3 2 is used to create the assay files for the KFmL KF96 KFflex96 and KFDuo instruments The respective assay file s can either be transferred onto the corresponding workstation or be started directly from within the Bindlt software after import However keep in mind that directly run assay files are not stored in the workstation memory Important Be advised that the Bindlt SW 3 2 uses a new type of file extension Therefore it is n
Download Pdf Manuals
Related Search