InviMag Universal Kit/ KFDuo User manual
Contents
1. 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 InviMag Universal Kit KFDuo w o plastic 0515 Important indications Preparing RNA When preparing RNA work quickly during the manual steps of the procedure Special care should be taken to avoid contaminations of reagents with DNases RNases Storing samples Frozen serum or plasma samples should not be thawed more than once Repeated freezing and thawing cycles may lead to denaturation and precipitation of proteins resulting in reduced titers and yields of nucleic acids Carrier RNA Carrier RNA serves two purposes It enhances the binding of nucleic acids to the beads especially if there are only very few target molecules in the sample Furthermore the addition of Carrier RNA reduces the chance of nucleic acid degradation in the rare event that RNase or DNase molecules are not inactivated completely by the Lysis Buffer Internal Controls The use of an internal control is recommended when using the InviMag Universal Kit KFDuo w o plastic in combination with diagnostic amplification systems Internal controls should be added directly to the lysis mixture during the pause step when the Binding Solution and beads are added Do not add any controls to the sample or to lysis mixture until the lysis step is complete In rare cases the controls may be degraded by2. End of step Washing Step I Beginning of step Mixing heating End of step Washing Step 2 Beginning of step Mixing heating End of step KingFisher Duo 12 tip comb Working Plate Working Plate Precollect Release beads Mixing time speed Heating temperature C Postmix Collect beads Post temperature Working Plate Message Dispensing volume ul Name Volume ul Name Volume ul Working Plate Precollect Release time speed Mixing time speed Heating during mixing Postmix Collect count Collect time s Post temperature Working Plate Precollect Release time speed Mixing time speed Heating during mixing Postmix Collect count Collect time s Post temperature Working Plate Precollect Release time speed Mixing time speed Heating during mixing Postmix Collect count Collect time s Post temperature 21 B Tip Comb A Lysis Binding No Yes 00 15 00 Medium 75 A Lysis Binding Add Isopropanol SNAPs 250 Isopropanol 230 SNAP Solution 20 A Lysis Binding No 00 00 10 Fast 00 05 00 Medium No No ot 5 No C Wash 1 No 00 00 10 Fast 00 05 00 Fast No D Wash 2 No 00 00 10 Fast 00 04 00 Fast No InviMag Universal Kit KFDuo w o plastic 0515 Washing Step 3 Beginning of step Mixing heating End of step Drying Step Elution Step Beginning of step Mixing heating
3. End of step Bead Removal Leave Working Plate Precollect Release time speed Mixing time speed Heating during mixing Postmix Collect count Collect time s Post temperature Working Plate Dry time Tip position Elution Stripe Precollect Release time speed Mixing time speed Heating temperature C Postmix Collect count Collect time s Post temperature Working Plate Release time speed Working Plate 22 Wash 3 No 00 00 10 Fast 00 03 00 Fast No No F 4 5 No Wash 3 00 05 00 Outside well tube A Elution No 00 00 10 Medium 00 10 00 Medium 65 No 4 10 No Wash 3 00 00 30 Fast B Tip Comb InviMag Universal Kit KFDuo w o plastic 0515 Troubleshooting Probable cause Comments and suggestions Low amount of extracted DNA Insufficient lysis Increase lysis time but prevent too long lysis time because this also decreases the yield Reduce amount of starting material Incomplete elution Increase the volume of Elution Buffer M ensure that the Elution Buffer M is transferred into the right position change the modified volume in the provided assay file too max 130 ul Low amount of SNAP Solution Mix SNAP Solution vigorously before use Too much Elution Buffer Elute the DNA in a lower volume of Elution Buffer M Change the modified volume in the run file too Low concentration of extracted DNA Incorrect storage of st
4. Stratecee molecular es User manual InviMag Universal Kit KFDuo for use on KingFisher Duo Thermo Fisher Scientific for automated purification of DNA genomic bacterial mitochondrial and viral as well as viral RNA from 200 ul clinical samples with magnetic beads 2450130X00 aaa STRATEC Molecular GmbH D 13125 Berlin dd Instruction for InviMag Universal Kit KFDuo w o plastic The InviMag Universal Kit KFDuo combines the advantages of the innovative InviMag technology with easy handling of magnetic particles in combination with the KingFisher Duo robotic platform from Thermo Fisher Scientific for a very efficient and reliable isolation of nucleic acids with a high purity The kit is the ideal tool for semi automated isolation and purification genomic and bacterial DNA and or viral DNA RNA from up to 200 ul sample volume The interplay of the nucleic acid extraction and purification chemistry provided by the InviMag Universal Kit KFDuo was intensely tested and validated The nucleic acid binding 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 high purity of the derived eluates the isolated nucleic acids are ready to use in a broad spectrum of downstream applications or can alternatively be stored at 20 C 80 C for subsequent use For research use only Tradema
5. 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 80 C This procedure minimizes degradation of crude DNA by limiting the activity of endogenous nucleases Storage of DNA Store DNA at 2 8 C Storage of genomic DNA at 20 C may cause shearing particularly if the DNA is exposed to repeated freezing and thawing cycles 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 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 shearing Avoid vigorous pipetting Pipetting genomic DNA through small tip openings can cause 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 and other small DNA Quantification Quantification of DNA and RNA from this a
6. DNases RNases present in the sample Alternatively internal controls can be prepared directly in the Carrier RNA tube In that case the amount of added water to the Carrier RNA has to be reduced Do not exceed the final filling volume of the Carrier RNA tube InviMag Universal Kit KFDuo w o plastic 0515 Scheme of the InviMag Universal 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 an unused cavity of row 1 of the 2 ml Deep Well Plate refers as Lysis Plate Add 20 ul Proteinase K and 20 ul Carrier RNA optional for genomic bacterial DNA preparations For bacterial DNA preparation 20 ul Lysozyme not provided stock solution 10 mg ml must be added Continue with the respective lysis protocol see pages 14 Prefill the Working Plate 2 ml DWP and Elution stripe with required buffers and appropriate volumes Working Plate 2 ml DWP Tip Row 2 Insert the KF Duo 12 Tip Comb in row 2 of the Working Plate Lysis Row 1 See lysis procedures page14 for respective protocol Wash 1 Row 3 Add 900 ul Wash Buffer HLT to row 3 of the Working Plate Wash 2 Row 4 Add 900 ul Wash Buffer M to row 4 of the Working Plate Wash 3 Row 5 Add 1000 ul Wash Buffer II to row 5 of the Working Plate Elution stripe Add 100 ul Elution Buffer M to the Elution stripe Please carefully read the protocols prior to the start of the prep
7. Plate before adding samples or other reagents Transfer 100 ul stool sample into a 2 ml tube and add 300 ul RNase free water Vortex the sample for 30 s followed by a 30 s centrifugation step at 3 000 rpm 1 000 x g Transfer 200 ul of the bacteria containing supernatant to a cavity of row 1 of the Working Plate Add 200 ul Lysis Buffer HLT and 20 ul Proteinase K Prefill the remaining rows of the Working Plate with required buffers and appropriate volumes and continue with the loading of the system see Starting a Run page 18 InviMag Universal Kit KFDuo w o plastic 0515 Starting a Run Preliminary Steps to process the sample onto the KingFisher System Important For working with the KFDuo instrument please carefully read the manufacturer s manual before using the system for the first time 1 Switch on the KF Duo instrument using the power switch 2 Prefill the Working Plate and Elution stripe with the required buffers and appropriate volumes Important Mix the bottle with the SNAP Solution by vigorously vortexing before usage Working Plate Lysis See the corresponding isolation protocol chapter Lysis procedures page 14 Tip Place the KF Duo 12 Tip Comb for DW magnets into row 2 of the Working Plate Wash 1 Add 900 ul Wash Buffer HLT to row 3 of the Working Plate Wash 2 Add 900 ul Wash Buffer M to row 4 of the Working Plate Wash 3 Add 1000 ul Wash Buffer Il to row 5 of the Working Plate Elution str
8. Universal Kit KFDuo w o plastic 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 a problem in the lot 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 Universal Kit KFDuo w o plastic 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 Universal Kit KFDuo w o plastic 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 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 InviMag Universal Kit KFDuo w o plastic 0515 Intended use The InviMag Universal Kit KFDuo w o plastic is designed for semi automated rapid and ec
9. 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 Supported MS Windows XP Pro with SP3 Windows Vista SP2 Windows 7 operating systems Disk space 500 MB free disk space Processor Intel Pentium gt 1 GHz Memor 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 Monitor color XVGA monitor with at least 1024x768 resolution and at least a 16 bit color settings 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 7 InviMag Universal Kit KFDuo w o plastic 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 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 work well in various
10. diagnostic assay should be interpreted with regards to other clinical or laboratory findings All utilities except ethanol and isopropanol required for preparation of nucleic acids are provided by the InviMag Universal Kit KFDuo w o plastic To minimize irregularities in diagnostic results adequate controls for downstream applications should be used Product use limitation The kit is validated for e g viral DNA RNA extraction from cell free body fluids and rinsed liquids specifically for human serum and plasma Related applications will need a separate validation Extraction of other than human DNA from blood or of total RNA has not been evaluated with this kit 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 products for any particular use STRATEC Molecular does not provide validations of performance characteristics of the product with respect to specific applications STRATEC Molecular products may be used in clinical diagnostic laboratory systems conditioned upon the complete diagnostic system of the laboratory The laboratory must be 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 q
11. of extracted RNA Low concentration of extracted RNA Degraded RNA RNA does not perform well in downstream applications e g real time RT PCR or RT PCR Eluted RNA is brownish colored Insufficient lysis Incomplete elution Low amount of SNAP Solution Too much Elution Buffer R Incorrect storage of starting material Incorrect Wash Buffers Incorrect storage of starting material Old material Ethanol carryover during elution Salt carryover during elution Small parts of the magnetic particles are left in the elution 24 Increase lyses time but prevent too long lyses time because this decrease the yield Reduce amount of starting material Use a higher volume of Elution Buffer M Ensure you pipet the Elution Buffer M with the correct volume to the right position Change the modified volume in the provided assay file too max 130 ul Mix SNAP Solution thoroughly before pipetting to the Deep Well Plate Elute the RNA with lower volume of Elution Buffer M Change the modified volume in the run file too 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 or isopropanol was added to the Wash Buffers ensure that the storage of starting material was correctly Avoid multiple thawing and freezing cyclesof the sample material Ensure that the starting material is fresh or stored a
12. F Deep Well Plate KF Duo 12 Tip Comb 8 x 12 preparations 2450130150 30 ml 2 pcs 2 pcs 2x 1 1 ml empty bottle final volume 30 ml 90 ml final volume 150 ml 45 ml final volume 150 ml 30 ml final volume 120 ml 15 ml 3x2ml 2 1 2 X 50 pcs Add 60 ml of abs Isopropanol to the bottle Wash Buffer HLT mix thoroughly and keep the bottle firmly closed Add 90 ml of 96 100 ethanol to the bottle Wash Buffer M mix thoroughly and keep the bottle firmly closed Add 105 ml of 96 100 ethanol to the bottle Wash Buffer II mix thoroughly and keep the bottle firmly closed Resuspend the Proteinase K in 1 1 ml RNase free water mix thoroughly until completely dissolving and store at 20 C Resuspend the Carrier RNA in 1 2 ml RNase free water Mix thoroughly until completely dissolving Add 30 ml 99 7 Isopropanol molecular biologic grade into the empty bottle 40 x 12 preparations 2450130250 120 ml 1 pcs 1 pcs 10 5 ml empty bottle final volume 120 ml 360 ml final volume 600 ml 180 ml final volume 600 ml 150 ml final volume 600 ml 60 ml 2x15 ml 10 1 10 x 50 pcs Add 240 ml of abs Isopropanol to the bottle Wash Buffer HLT mix thoroughly and keep the bottle firmly closed Add 450 ml of 96 100 ethanol to the bottle Wash Buffer M mix thoroughly and keep the bottle firmly closed Add 420 ml of 96 100 ethanol to the bottle Wash Buffer II mix thoroug
13. For the preparation of bacterial DNA 20 ul Lysozyme stock 10 mg ml not provided have to be added for an improved lysis Add the Lysozyme directly to row 1 of the Working Plate before adding samples or other reagents Transfer 200 ul of the rinsed liquid into a free cavity of row 1 of the Working Plate and add 20 ul Proteinase K and 20 ul Carrier RNA If genomic or bacterial DNA is prepared the addition of Carrier RNA is optional Prefill the remaining rows of the Working Plate with the required buffers and appropriate volumes and continue with the loading of the system see Starting a Run page 18 InviMag Universal Kit KFDuo w o plastic 0515 b the sample will not be used for cultivation Cut off the relevant part of the swab and transfer it into an RNase and DNAse free 2 ml tube Add 300 ul RNase free water to the swab and vortex intensely for 3 min Optional incubate for 3 min at 95 C For the preparation of bacterial DNA 20 ul Lysozyme stock 10 mg ml not provided should be used for an improved lysis Add the Lysozyme directly to row 1 of the Working Plate before adding samples or other reagents Transfer 200 ul of the rinsed liquid into a cavity of row 1 of the Working Plate and add 20 ul Proteinase K and 20 ul Carrier RNA If genomic or bacterial DNA is prepared the addition of Carrier RNA is optional Prefill all remaining rows of the Working Plate with the required buffers and appropriate volumes and continue with t
14. al safety regulations European Community risk and safety phrases for the components of the InviMag Universal Kit KFDuo w o plastic to which they apply are listed below as follows Lysis Buffer HLT Proteinase K contains guanidine hydrochloride warning danger H302 315 319 P280 505 351 338 H315 319 334 335 P280 305 351 338 310 405 Wash Buffer HLT contains guanidine hydrochloride warning H302 315 319 P280 505 351 338 H302 Harmful if swallowed 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 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 InviMag Universal Kit KFDuo w o plastic 0515 Product characteristics of the InviMag Universal Kit KFDuo w o plastic preparation up to 200 ul cell free body fluids like Depends on sample storage and source AERO serum plasma or liquor etc Note The added Carrier RNA will account for up to 200 ul rinsed liquid from swabs most of the e
15. aration 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 20 ul SNAP Solution have to be added to each lysate in row 1 of the Working Plate Important If an internal extraction control should be used please add it to the reaction mixture at this step Nucleic acids bind to magnetic particles Washing of the particle fixed nucleic acids Magnetic separation Elution of nucleic acids Magnetic Separation Pure nucleic acids InviMag Universal Kit KFDuo w o plastic 0515 Lysis procedures For easier handling we recommend to prepare a master mix consisting of Lysis Buffer HLT Proteinase K and if required Carrier RNA When preparing the Master Mix it is recommended to prepare a volume of 5 greater than that required Protocol 1 Simultaneous isolation of nucleic acids viral DNA RNA from cell free body fluids or of DNA from blood genomic DNA Please read the protocols carefully prior to the start of the preparation procedure Important Note The protocol is optimized for a sample volume of 200 ul For smaller samples volumes than 200 ul please adjust to a total volume of 200 ul with ddH2O or PBS Transfer 200 ul of sample into a free cavity of row 1 of the Working Plate and add 200 ul Lysis Buffer HLT 20 ul Proteinase K and 20 ul Carrier RNA If genomic DNA shall be prepared the addition of Carrier RNA is opti
16. arting Ensure that the storage of starting material material was correct Avoid repeated thawing and freezing cycles of the sample material Incorrect Wash Buffers Ensure that the correct amount of ethanol or isopropanol was added to the Wash Buffers and they are stored correctly Degraded DNA Incorrect storage of starting Ensure that the storage of starting material material was correct Old material Ensure that the starting material is stored at appropriate conditions 20 C 80 C avoid multiple thawing and freezing cycles of the material DNA does not perform No PCR result for genomic DNA Due to the very gentle isolation procedure well in downstream it may happen that isolated genomic DNA applications e g real time forms a ball To overcome this the PCR or PCR primary PCR denaturation step at 95 C should be prolonged to 5 min Increase drying time for removal of Ethanol carryover during elution ethanol in the assay file Salt carry over during elution 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 equilibrated at room temperature Eluted DNA is brownish Small part of the magnetic Centrifuge at full speed for 1 min and colored particles are left in the elution transfer supernatant to a new tube j5 InviMag Universal Kit KFDuo w o plastic 0515 Probable cause Comments and suggestions Low amount
17. ation Transportation of the SNAP bound nucleic acids into Wash 1 3 First Washing Step Sample washing for 5 min SNAP separation Transportation of the SNAP bound nucleic acids into the Wash 2 4 Second Washing Step Sample washing for 4 min SNAP separation Transportation of the SNAP bound nucleic acids into the Wash 3 5 Third Washing and Drying Step Sample washing for 3 min SNAP separation Drying the SNAP bound nucleic acids outside of the plate for 5 min Transportation of the SNAP into the Elution stripe 6 Elution of the nucleic acids Incubation of the SNAP bound nucleic acids into the Elution stripe for 10 min by mixing at elevated temperature SNAP separation The SNAPs without the bound nucleic acids are afterwards automatically discarded into the wells of Wash 3 row 5 of the Working Plate disposal Important Notes After finishing the extraction protocol the Elution stripe contains the extracted nucleic acids Store the nucleic acids at adequate conditions We recommend transferring the extracted nucleic acids into 1 5 ml Receiver Tubes provided and store them at 20 C or 80 C If the extracted nucleic acids contain carry over of magnetic particles transfer them into a 1 5 ml reaction tube centrifuge at max speed for 1 min and then transfer the nucleic acids containing supernatant into the provided Receiver Tubes The eluted nucleic acids are ready to use in different downstream applications InviMag Univ
18. col is not validated for the isolation of DNA from swabs using storage buffers from other providers As long as the samples are not shock frosted in liquid nitrogen or incubated with RNase inhibitors or denaturing reagents the viral RNA is not secured Therefore it is essential that samples are immediately flash frozen subsequent to harvesting by using liquid nitrogen and storage at 80 C Viral RNA in deep frozen samples is stable for months Viral RNA purification should be processed as soon as possible Urine The bacteria should be pelleted while the supernatant is discarded urea contaminations may inhibit PCR reactions For some applications fresh urine can be used directly Blood Best results are obtained using fresh blood samples Blood samples stabilized with EDTA or citrate but not heparin can be stored at room temperature 18 25 C for 2 3 hours For short term storage up to 24h samples should be stored at 4 8 C For long term storage we recommend to freeze the samples at 20 C or 80 C Avoid multiple thawing and freezing cycles of the sample s before isolating the DNA RNA because this may lead to degraded DNA Stool samples Best results are obtained with fresh material Stool samples contain DNases and RNases which realize quickly DNA and RNA digestion and degradation The sample may be stored frozen at 80 C Serum and plasma and other cell free body fluids After collection and centrifugation serum or plasma der
19. e 6 Do not use damaged kit components because their use may lead to poor kit performance o Always change pipet tips between liquid transfers We recommend the use of aerosol barrier pipet tips to avoid cross contaminations 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 immediately Do not combine components of different kits unless the lot numbers are identical Avoid microbial contamination of the kit reagents We recommend working under laminar air flow until the samples are lysed to minimize the risk of infections from potentially infectious material O O O O O This kit should only be used by trained personnel InviMag Universal Kit KFDuo w o plastic 0515 Preparing reagents and buffers 8 x 12 extractions Add 60 ml of abs Isopropanol to the bottle Wash Buffer HLT mix thoroughly and keep the bottle firmly closed Add 90 ml of 96 100 ethanol to the bottle Wash Buffer M mix thoroughly and keep the bottle firmly closed Add 105 ml of 96 100 ethanol to the bottle Wash Buffer Il mix thoroughly and keep the bottle firmly closed Resuspend the Proteinase K in 1 1 ml RNase free water mix thoroughly until completely dissolving and store at 20 C Resuspend the Carrier RNA in 1 2 ml RNase free water Mix thoroughly until completely dissolving Add 30 ml of 99 7 Isopropano
20. ersal Kit KFDuo w o plastic 0515 Reagent info A Lysis Binding Name Sample Lysis Buffer HLT Proteinase K Carrier RNA if required Lysozyme if required B Tip Comb Name C Wash 1 Name Wash Buffer HLT D Wash 2 Name Wash Buffer M E Wash 3 Name Wash Buffer H Name Name Name A Elution Name Elution Buffer M Dispensed reagents A Lysis Binding Well volume ul 200 200 20 50 20 Well volume ul Well volume ul 900 Well volume ul 900 Well volume ul 1000 Well volume ul Well volume ul Well volume ul Well volume ul 100 20 For self programming of the KFDuo instrument Working Plate Total reagent volume ul Working Plate Total reagent volume ul Working Plate Total reagent volume ul Working Plate Total reagent volume ul Working Plate Total reagent volume ul Working Plate Total reagent volume ul Working Plate Total reagent volume ul Working Plate Total reagent volume ul Hution Stripe Total reagent volume ul Working Plate Type Sample Reagent Reagent Reagent Reagent Type Type Reagent Type Sample Type Reagent Type Type Type Type Reagent InviMag Universal Kit KFDuo w o plastic 0515 Steps data Tip 1 p Pick Up Lysis Step Beginning of step Mixing heating End of step Adjust Binding Reagent s Binding Step Beginning of step Mixing heating
21. he loading of the system see Starting a Run page 18 Protocol 3 Simultaneous isolation of nucleic acids DNA and RNA from tissue biopsies Please read the protocols carefully prior to the start of the preparation procedure Transfer 1 10 mg from the tissue biopsy sample into a cavity of row 1 of the Working Plate and add 200 ul distilled water 200 ul Lysis Buffer HLT 20 ul Carrier RNA and 20 ul Proteinase K If genomic DNA is prepared the addition of Carrier RNA is optional For the preparation of bacterial DNA 20 ul Lysozyme stock 10 mg ml not provided should be added for an improved lysis Prefill all remaining rows of the Working Plate with the required buffers and appropriate volumes and continue with the loading of the system see Starting a Run page 18 Protocol 4 Isolation of DNA from bacteria pellets up to 1x 10 bacterial cells or from some ml of non viscous tracheal secrete of BAL Please read the protocols carefully prior to the start of the preparation procedure For the preparation of bacterial DNA we recommend using 20 ul Lysozyme stock 10 mg ml not provided for an improved lysis Add the Lysozyme directly to row 1 of the Working Plate before adding samples or other reagents For sample preparation use an aliquot from the bacterial culture and centrifuge the sample at 9 300 x g 10 000 rpm for 3 min Discard the supernatant without disturbing the bacterial pellet Resuspend the bacterial pellet
22. hly and keep the bottle firmly closed Resuspend the Carrier RNA in 15 ml RNase free water Mix thoroughly until completely dissolving Resuspend the Proteinase K in 10 ml RNase free water mix thoroughly until completely dissolving and store at 20 C Add 120ml of 99 7 Isopropanol molecular biologic grade into the empty bottle see order information KF Duo Elution stripe InviMag Universal Kit KFDuo w o plastic 0515 Symbols Lo Lot number Catalogue number Expiry date Consult operating instructions Temperature limitation Os BRIA Do not reuse Storage All buffers and kit contents of the InviMag Universal Kit KFDuo w o plastic except Carrier RNA and SNAP solution should be stored at room temperature and are stable for at least 12 months at this condition Carrier RNA Proteinase K Lyophilized Carrier RNA must be stored at 2 8 C Dissolved Carrier RNA and Proteinase K must be stored at 20 C SNAP solution The magnetic beads should be stored at 2 8 C Wash Buffer M Wash Buffer Il Wash Buffer HLT Wash Buffers charged with either ethanol or isopropanol should be stored at room temperature and must be appropriately sealed 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
23. in 200 ul distilled water or PBS and transfer the sample into a cavity of row 1 of the Working Plate Add 200 ul Lysis Buffer HLT and 20 ul Proteinase K Prefill all remaining rows of the Working Plate with the required buffers and appropriate volumes and continue with the loading of the system see Starting a Run page 18 InviMag Universal Kit KFDuo w o plastic 0515 Protocol 5 Simultaneous isolation of total nucleic acids from sputum Please read the protocols carefully prior to the start of the preparation procedure For the preparation of bacterial DNA we recommend using 20 ul Lysozyme stock 10 mg ml not provided for an improved lysis Add the Lysozyme directly to row 1 of the Working Plate before adding samples or other reagents Transfer 150 ul of the soutum sample into an RNase DNAse free tube and add 150 ul NAC Buffer order number 1033221100 or saturated acetylcysteine ACC solution to the sample ratio sample to buffer must be 1 1 Incubate the mixture for 10 min at 95 C to reduce the viscosity and transfer 200 ul from the mixture into a free cavity of row 1 of the Working Plate Add 200 ul Lysis Buffer HLT 20 ul Carrier RNA and 20 ul Proteinase K If genomic or bacterial DNA is processed the addition of Carrier RNA is optional Prefill all remaining rows of the Working Plate with the required buffers and appropriate volumes and continue with the loading of the system see Starting a Run page 18 Pr
24. ipe Add 100 ul Elution Buffer M to the separate elution stripe 3 Choose the assay InviMag Universal KF Duo on the display of the instrument and press the START button 4 Insert the prefilled Working Plate and Elution stripe onto the corresponding positions of the KingFisher Duo surface Press the START button to initialize the assay 5 After the lysis steps a pause will occur and 230 ul Binding Solution and 20 ul SNAP Solution have to be added to each used cavity of row 1 of the Working Plate If extraction controls should be used please add them at this step too Alternatively the internal control can be added to the Carrier RNA tube see page 12 6 Reinsert the Working Plate into the instrument watch out for correct plate orientation and continue the run by pressing the START button The instrument will now finish the purification process without any further user interaction InviMag Universal Kit KFDuo w o plastic 0515 ll The following steps run automatically on the KingFisher System Specific sample preparation 1 Lysis Lysis is performed at elevated temperature for 15 min After lysis the instrument will be paused and the user has to add 230 ul Binding Solution and 20 pl SNAP Solution magnetic beads Internal extraction controls should be added during the pause step too or can alternatively be added to the Carrier RNA see page 12 2 Binding of the nucleic acids Binding step for 5 min SNAP separ
25. ived from blood treated with anticoagulants like EDTA or citrate but not with heparin synovial fluid samples or other cell free body fluids and rinsed liquids from swabs can be used for extraction For short term storage samples can be kept on ice for 1 2 hours or for up to 24 hours samples may be stored at 20 C For long term storage we recommend freezing samples in aliquots at 80 C Frozen plasma or serum samples must not be thawed more than once Multiple thawing and freezing cycles before isolating the viral DNA RNA should be avoided because this may lead to denaturation precipitation of proteins resulting in reduced viral titers and therefore reduced yields of viral nucleic acids In addition cryoprecipitate formed during freeze thawing cycles can cause problems If cryoprecipitate are visible pellet them by centrifugation at 6 800 x g for 3 minutes The cleared supernatant should be aspirated without disturbing the pellet and processed immediately This step will not reduce viral titers Cell culture supernatants Best results are obtained with fresh material or material that has been immediately frozen and stored at 20 C or 80 C after obtaining the cell culture supernatant Repeated freezing and thawing cycles of stored samples will negatively influence the sensitivity STRATEC Molecular will be released of its responsibilities if other sample materials than described in the chapter Intended Use are processed or if the samp
26. l molecular biologic grade into the empty bottle 40 x 12 extractions Add 240 ml of abs Ilsopropanol to the bottle Wash Buffer HLT mix thoroughly and keep the bottle firmly closed Add 450 ml of 96 100 ethanol to the bottle Wash Buffer M mix thoroughly and keep the bottle firmly closed Add 420 ml of 96 100 ethanol to the bottle Wash Buffer Il mix thoroughly and keep the bottle firmly closed Resuspend the Carrier RNA in 15 ml RNase free water Mix thoroughly until completely dissolving Resuspend the Proteinase K in 10 ml RNase free water mix thoroughly until completely dissolving and store at 20 C Add 120 ml of 99 7 Isopropanol molecular biologic grade into the empty bottle Reagents and equipment to be supplied by user When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information s please consult the appropriate material safety data sheets MSDS on our webpage www stratec com Microcentrifuge gt 9 300 x g 10 000 rpm optional Ethanol 96 100 1 5 ml reaction tubes optional Measuring cylinder 250 ml Disposable gloves Pipet with tips we highly recommend to use filter tips only 15 or 50 ml reaction tubes optional lsopropanol O O O O 0 O The InviMag Universal 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
27. l and allowed to air dry o Non disposable plastic ware must be treated before use to ensure that it is RNase free Plastics should be thoroughly rinsed with 0 1 M NaOH 1 mM EDTA followed by RNase free water It is possible to use chloroform resistant plastic ware rinsed with chloroform to inactivate RNases All solutions must be prepared with DEPC treated RNase free water Change gloves frequently and keep tubes closed When handling RNA reduce the preparation time as much as possible Only use sterile disposable polypropylene tubes throughout the procedure Always keep RNA samples on ice O O O 0 This kit should only be used by personnel trained in laboratory practice Storage of RNA Purified viral RNA can be stored at 80 C and is stable for several years at this condition Quantification Quantification of DNA and RNA from this assay must be done by means of qPCR or Reverse Transcriptase qPCR All other methods will be disturoed by the included Carrier RNA as well as DNA or RNA which is co purified m InviMag Universal Kit KFDuo w o plastic 0515 Ordering information Product Package Size Catalogue No InviMag Universal Kit KFDuo w o plastic 8 x 12 preparations 2450130150 InviMag Universal Kit KFDuo w o plastic 40 x 12 preparations 2450130250 KingFisher Duo and consumables KingFisher Duo 5400100 KingFisher Duo 12 tip comb 50 pieces 5012501000 KingFisher Duo elution strip 40 pieces 9012501100 DeepWell pla
28. le preparation protocols are changed or modified InviMag Universal Kit KFDuo w o plastic 0515 Principle and procedure The InviMag Universal Kit KFDuo w o plastic procedure comprises following steps Sample preparation if required Lysis at different temperatures Adjustment of binding conditions Binding of the nucleic acids to magnetic beads Washing of the bead bound nucleic acids and evaporation of ethanol Elution of nucleic acids O O O 000 Procedure Bacteria must be cultivated at special conditions An aliquot of the bacterial suspension is used to form a pellet by centrifugation at high speed for 5 min The supernatant is discarded Pretreatment Please check the corresponding section in the protocol Lysis Samples are lysed at elevated temperatures in the presence of Lysis Buffer HLT and Proteinase K Lysozyme if required to break bacterial and viral cell walls and to digest proteins The addition of Carrier RNA is required for the enhancement and stabilization of viral DNA RNA recovery Due to this it is even possible to purify very small amounts of viral DNA RNA molecules Binding of the nucleic acids After addition of Binding Solution to adjust optimal binding conditions the nucleic acids are bound by the simultaneously added magnetic beads SNAP Solution Removing residual contaminants Contaminants are efficiently removed during the washing process using Wash Buffer HLT Wash Buffer M and Wash Buffer II
29. liers for lsopropanol Carl Roth Applichem Sigma 2 Propanol 2 Propanol 2 Propanol Rotipuran gt 99 7 p a ACS ISO Order no A3928 Order no 59304 1L F Order no 6752 Possible suppliers for centrifuges Eppendorf AG SIGMA Laborzentrifugen GmbH 22331 Hamburg Germany 37507 Osterode am Harz Germany Phone 49 0 40 53801 0 Phone 49 5522 5007 0 Fax 49 0 40 53801 556 Fax 49 5522 5007 12 E Mail eppendorf eppendorf com E Mail info sigma zentrifugen de Internet www eppendorf com Internet www sigma zentrifugen de j InviMag Universal Kit KFDuo w o plastic 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 1G7009 05 2015
30. ltaneous isolation of nucleic acids DNA RNA from swab material 14 Protocol 3 Simultaneous isolation of nucleic acids DNA and RNA from tissue biopsies15 Protocol 4 Isolation of DNA from bacteria pellets up to 1x 10 bacterial cells 15 Protocol 5 Simultaneous isolation of total nucleic acids from sputum 16 Protocol 6 Simultaneous isolation of total nucleic acids DNA and RNA from tracheal secretes or BAL 16 Protocol 7 Simultaneous isolation of viral nucleic acids from stool samples 17 Protocol 8 Isolation of bacterial DNA from stool samples 17 Starting a Run 18 For self programming of the KFDuo instrument 20 Troubleshooting 23 Appendix 25 General notes on handling DNA 26 General notes on handling RNA 27 Ordering information 28 InviMag Universal Kit KFDuo w o plastic 0515 Kit contents of InviMag Universal Kit KFDuo w o plastic Store the SNAP Solution and Carrier RNA at 2 8 C Store dissolved Proteinase K Carrier RNA in aliquots at 20 C Store all other kit components at room temperature RT Important The needed KFDuo plastic is not included in the kit see ordering information at page 28 Component Catalogue No Lysis Buffer HLT Carrier RNA Proteinase K SNAP Solution Binding Solution fill with 99 7 Isopropanol Wash Buffer HLT Wash Buffer Il Wash Buffer M Elution Buffer M RNase free water Sealing Foils Manual 1 5 ml Receiver Tubes Initial steps Plastic to be supplied by user 2 0 ml K
31. luted nucleic acids up to 200 ul cell culture supernatants Quantitative RT PCR is recommended for up to 200 ul blood EDTA citrate determination of the viral RNA or DNA yield stabilized but not heparin 1x 10 mammalian cells 10 mg tissue sample The InviMag Universal Kit KFDuo w o plastic is the ideal tool for an efficient and semi automated extraction of genomic and or bacterial DNA and viral DNA RNA from different sample sources The nucleic acid isolation process is based on the interaction of nucleic acids with silica coated magnetic particles in presence of adjusted buffer conditions The DNA RNA purification procedure is performed with minimal user intervention except the initial loading of the system and plate preparation This allows safe handling of potentially infectious samples Sample cross contamination and reagent cross over is effectively eliminated The KingFisher instrument uses magnetic rods to transport the DNA RNA binding magnetic particles through the various extraction phases lysis binding washing elution The automated purification process results in a fast reliable and robust technique After a sample specific lysis optimal binding conditions are adjusted The genomic DNA and or viral DNA RNA will bind to the added magnetic particles and is separated from the solution by magnetic rods controlled by the KingFisher system Subsequent to three washing steps the nucleic acids are finally eluted fro
32. m the beads Due to the high purity the eluted nucleic acids are ready to use in a broad panel of downstream applications like PCR real time PCR Restriction Enzyme Digestion HLA Typing o Southern Blot O O O 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 Blood Mini 96 HTS Kits for use on a centrifuge vacuum manifold or other robotic stations For the isolation of viral RNA DNA or both STRATEC Molecular offers a series of spin kits as well as HTS kits for use on centrifuge vacuum manifold or for a walk away automated isolation on other robotic stations as well as magnetic bead based kits See page 28 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 Universal Kit KFDuo w o plastic 0515 Sampling and storage of starting material For reproducible and high yields the correct sample storage is essential Yields may vary from sample to sample depending on factors such as health of the donor sample age kind of sample transport and storage conditions Best results are obtained with fresh material Cultivated bacteria or bacterial suspension s Bacteria have to be pelleted after cultivation and need to be resuspended at defined conditions Swabs Saliva The protocol works with fresh saliva prepared swabs as well as with dried swabs The proto
33. onal Prefill the remaining rows of the Working Plate with the required buffers and appropriate volumes and continue with the loading of the system see Starting a Run page 18 Protocol 2 Simultaneous isolation of nucleic acids DNA RNA from swab material Please read the protocols carefully prior to the start of the preparation procedure 2 1 Use of swabs Transfer 200 ul Lysis Buffer HLT into a cavity of row 1 of the Working Plate and add 200 ul distilled water 20 ul Proteinase K and 20 ul Carrier RNA If bacterial DNA is processed 20 ul Lysozyme not provided stock solution 10 mg ml should be added If genomic or bacterial DNA is processed the addition of Carrier RNA is optional Insert the swab into the cavity of row 1 of the Working Plate Incubate for 5 10 min at RT and stir occasionally After incubation remove the swab and squeeze it out inside the corresponding cavity to remove residual liquid and then discard the swab Prefill the remaining rows of the Working Plate with the required butfers and appropriate volumes and continue with the loading of the system see Starting a Run page 18 2 2 Usage of rinsed liquid from swab a the sample will also be used for cultivation Cut off the relevant part of the swab and transfer it into an RNase DNAse free 2 ml tube Add 300 ul physiological saline solutions to the swab and vortex intensely for 2 3 min and incubate for 10 min at RT Use an aliquot for cultivation
34. onomical preparation of nucleic acids viral DNA RNA genomic DNA bacterial DNA but not plasmid DNA from 200 ul sample volumes like blood EDTA Citrate stabilized but not heparin serum plasma cerebrospinal fluid cell culture supernatant cell free body fluids urine supernatant from stool suspensions rinsed liquid from swabs or bacterial suspensions sputum BAL using magnetic beads and the KingFisher Duo instrument from Thermo Fisher Scientific The whole process is based on the patented InviMag technology which relies on binding of the nucleic acids by magnetic particles The procedure only requires minimal user interaction prefilling of reagents allowing safe handling of potentially infectious samples The provided isolation protocols and buffers are optimized to provide high yields and purities However for reproducible yields an appropriate sample storage and quick handling is essential The purified viral DNA and or RNA as well as bacterial or genomic DNA are ready to use for downstream analysis THE PRODUCT IS INDENTED FOR USE BY PROFESSIONAL WORKERS SUCH AS TECHNICIANS PHYSICIANS AND BIOLOGISTS TRAINED IN MOLECULAR BIOLOGICAL TECHNIQUES The generated eluates can be used with any downstream application employing enzymatic amplification or other modifications of DNA RNA followed by signal detection or amplification Any diagnostic results generated using the sample preparation procedure in conjunction with any downstream
35. otocol 6 Simultaneous isolation of viral nucleic acids DNA and RNA from slimy tracheal secretes or BAL Please read the protocols carefully prior to the start of the preparation procedure Important For the preparation of bacterial DNA we recommend using 20 ul Lysozyme stock 10 mg ml not provided for an improved lysis Add the Lysozyme directly to row 1 of the 2 ml DWP refers as Working Plate before adding samples or other reagents Non viscous samples Transfer 1 ml of tracheal secret or BAL into an RNase DNAse free tube and centrifuge at 9 300 x g 10 000 rpm for 3 min Discard the supernatant without disturbing the bacterial pellet Resuspend the bacterial pellet in 200 ul distilled water or PBS and transfer the sample into row 1 of the Working Plate Add 200 ul Lysis Buffer HLT 20 ul Carrier RNA and 20 ul Proteinase K Prefill all remaining rows of the Working Plate with the required buffers and appropriate volumes and continue with the loading of the system see Starting a Run page 18 Viscous sample Transfer 1 ml of tracheal secrete or BAL into a RNase and DNAse free tube and add 1 ml NAC Buffer order number 1033221100 or saturated acetylcysteine ACC solution to the sample ratio sample to buffer must be 1 1 Incubate the mixture for 10 min at 95 C to reduce the viscosity and centrifuge at 9 300 x g 10 000 rom for 3 min Discard the supernatant without disturbing the bacterial pellet directly Resus
36. pend the bacterial pellet in 200 ul distilled water or PBS and transfer to a free cavity of row 1 of the Working Plate Add 200 ul Lysis Buffer HLT 20 ul Carrier RNA and 20 ul Proteinase K Prefill all remaining rows of the Working Plate with the required buffers and appropriate volumes and continue with the loading of the system see Starting a Run page 18 InviMag Universal Kit KFDuo w o plastic 0515 Protocol 7 Simultaneous Isolation of viral nucleic acids from stool samples Please read the protocols carefully prior to the start of the preparation procedure Transfer 100 ul of stool sample into a 2 ml tube and dilute the sample 1 10 with RNase free water Vortex the sample for 30 s followed by a 1 min centrifugation step at 12 000 x g 13 000 rpm Transfer 200 ul of viral containing supernatant to row 1 of the Working Plate Add 200 ul Lysis Buffer HLT 20 ul Carrier RNA and 20 ul Proteinase K to each sample containing cavity Prefill the remaining rows of the Working Plate with the required buffers and appropriate volumes and continue with the loading of the system see Starting a Run page 18 Protocol 8 Isolation of bacterial DNA from stool samples Please read the protocols carefully prior to the start of the preparation procedure Important For the preparation of bacterial DNA we recommend using 20 ul Lysozyme stock 10 mg ml not provided for an improved lysis Add the Lysozyme directly to row 1 of the Working
37. rks 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 Universal Kit KFDuo w o plastic 0515 Table of content Kit contents of InviMag Universal Kit KFDuo w o plastic 3 Symbols 4 Storage 4 Quality Control and product warranty 4 Intended use gt Product use limitation 5 Safety information 6 Product characteristics of the InviMag Universal Kit KFDuo 7 Sampling and storage of starting material 8 Principle and procedure 9 Procedure 9 Yield and quality of genomic DNA derived from Blood 9 Yield and quality of viral nucleic acids 10 Important notes 10 Preparing reagents and buffers 11 Reagents and equipment to be supplied by user 11 Important indications 12 Scheme of the InviMag Universal Kit KFDuo w o plastic 13 Lysis procedures 14 Protocol 1 Simultaneous isolation of nucleic acids viral DNA RNA from cell free body fluids or blood genomic DNA 14 Protocol 2 Simu
38. s vary in efficiency depending on the total amount of nucleic acid present in the reaction Eluates derived by this kit will contain Carrier RNA which will greatly exceed the amount of the isolated viral NA Yields of viral nucleic acids isolated from biological samples are usually low concentrated and therefore almost impossible to determine photometrically Keep in mind that the Carrier RNA 5 ug per 200 ul sample will account for most of the present RNA The kit is suitable for downstream analysis with NAT techniques for examples qPCR RT qPCR LAMP LCR Diagnostic assays should be performed accordingly to the manufacturer s instructions Quantitative RT PCR is recommended for determination of viral RNA yield In Gel Electrophoresis and in Capillary Electrophoresis RNA extracted with the provided kit looks like degraded cause the kit contains Carrier RNA this is poly A RNA in fragments of 100 up to 1000 bases The kit is not dedicated for applications using this kind of analysis Important notes 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 pag
39. ssay must be done by means of qPCR or Reverse Transcriptase qPCR All other methods will be disturbed by the included Carrier RNA as well as DNA or RNA which will be co purified sp InviMag Universal Kit KFDuo w o plastic 0515 General notes on handling RNA RNA is far less stable than DNA It is very sensitive to degradation by endogenous RNases in the biological material and exogenous RNases which are permanently present everywhere in the lab To achieve satisfactory qualitative and quantitative results in RNA preparations contaminations with exogenous RNases have to be reduced to a minimum Avoid handling bacterial cultures cell cultures or other biological sources of RNases in the same lab where the RNA purification is carried out All glassware must be RNase free Therefore the glassware should be cleaned with detergent thoroughly rinsed and oven baked at 240 C for four or more hours before use Autoclaving only will not completely inactivate many RNases Oven baking will inactivate RNases and ensure that no other nucleic acids such as plasmid DNA are present on the surface of the glassware It is possible to clean glassware with 0 1 DEPC diethyl pyrocarbonate The glassware must react 12 hours at 37 C and should then be autoclaved or heated to 100 C for 15 min to inactivate residual DEPC o Electrophoresis tanks should be cleaned with detergent solution e g 0 5 SDS thoroughly rinsed with RNase free water rinsed with ethano
40. t appropriate conditions 20 C 80 C Avoid multiple thawing and freezing of the material increase drying time for removal of ethanol in the assay file 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 equilibrated at room temperature Centrifuge at full soeed for 1 min and transfer supernatant into a new tube InviMag Universal Kit KFDuo w o plastic 0515 Appendix KingFisher Bindlt Software 3 2 or higher versions Bindlt software 3 2 or higher versions was used to create the assay file s for the KFmL KF96 KFflex96 or KFDuo 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
41. te 2 ml KingFisher 50 pieces 5012401700 Related Products Package Size Catalogue No InviMag Pathogen Kit KF96 1 x 96 preparations 7450300100 InviMag Pathogen Kit KF96 5 x 96 preparations 7450300200 RTP Pathogen Kit 50 preparations 1040500200 RTP Pathogen Kit 250 preparations 1040500300 RTP DNA RNA Virus Mini Kit 50 preparations 1040100200 RTP DNA RNA Virus Mini Kit 250 preparations 1040100300 RTP Bacteria DNA Mini Kit 50 preparations 1033200200 RTP Bacteria DNA Mini Kit 250 preparations 1033200300 Invisorb Virus RNA HTS 96 Kit X 4 x 96 preparations 7143310300 Invisorb Virus RNA HTS 96 Kit X 24 x 96 preparations 7143310400 Invisorb Virus DNA HTS 96 Kit X 4 x 96 preparations 7142310300 Invisorb Virus DNA HTS 96 Kit X 24 x 96 preparations 7142310400 InviMag Virus RNA Mini Kit KF 96 1 x 96 preparations 7443300100 InviMag Virus RNA Mini Kit KF 96 5 x 96 preparations 7443300200 InviMag Virus DNA Mini Kit KF 96 1 x 96 preparations 7442300100 InviMag Virus DNA Mini Kit KF 96 5 x 96 preparations 7442300200 InviMag Virus DNA RNA Mini Kit KFmL 15 preparations 2441150100 InviMag Virus DNA RNA Mini Kit KFmL 75 preparations 2441150200 InviMag Virus DNA RNA Mini Kit 50 preparations 1440100200 InviMag Virus DNA RNA Mini Kit 250 preparations 1440100300 InviMag Bacteria DNA Mini Kit KFmL 15 preparations 2433150100 InviMag Bacteria DNA Mini Kit KFmL 75 preparations 2433150200 Possible supp
42. uality 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 The chemicals and plastic parts are for laboratory use only They should be stored in the laboratory and must not be used for other purposes than intended The kit components are not suitable for consumption InviMag Universal 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 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 Universal 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 must be considered and handled as infectious and discarded accordingly to loc
43. while the nucleic acids remain bound to the beads Elution The nucleic acids are finally eluted in Elution Buffer M and are ready to use in different subsequent downstream applications e g PCR amplification digestion with restriction enzymes Southern hybridizations HLA typing etc Yield and quality of genomic DNA derived from Blood The amount of purified DNA RNA in the InviMag Universal Kit KFDuo procedure from whole blood depends on the leucocytes content the sample source transport storage and age Typically a volume of 200 ul whole blood from a healthy individual with an elevated white blood cell content ranging from 3x10 to 1x10 cells ml will yield 2 6 ug of genomic DNA If a whole blood sample is mixed with anticoagulant containing buffer solutions the overall leukocyte concentration decreases and the yield is reduced Please keep in mind that the added Carrier RNA will falsify the real genomic DNA content in photometric measurements InviMag Universal Kit KFDuo w o plastic 0515 Yield and quality of viral nucleic acids The amount of purified nucleic acids in the InviMag Universal Kit KFDuo w o plastic procedure depends on the sample type virus titer sample source transport storage and age Yield and quality of the isolated viral nucleic acids is suitable for any detection system Diagnostic tests should be performed accordingly to the manufacturers specifications Different amplification system
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