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InviMag Universal Kit/ KF96 User manual

1. Dry time Tip position Elution Plate Precollect Release time speed Mixing time speed Heating temperature C Preheat Postmix Collect count Collect time s Wash Plate 3 Release time speed Tip Plate No 00 00 10 Fast 00 03 00 Fast UI py 7 O O 00 05 00 Outside well tube No 00 00 10 Medium 00 10 00 Medium 00 00 30 Fast InviMag Universal Kit KF96 0515 Troubleshooting Probable cause Comments and suggestions Low amount of extracted DNA Low concentration of extracted DNA Degraded DNA DNA does not perform well in downstream applications e g real time PCR or PCR Eluted DNA is brownish colored Insufficient lysis Incomplete elution Low amount of SNAP Solution Too much Elution Buffer Incorrect storage of starting material Incorrect Wash Buffers Incorrect storage of starting material Old material No PCR result for genomic DNA Ethanol carryover during elution Salt carry over during elution Small part of the magnetic particles are left in the elution 28 Increase lysis time but prevent too long lysis time because this also decreases the yield Reduce amount of starting material 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 Mix SNAP Solution vigorously before use Elute the DNA with in a lower volume of E
2. 1 29 2507 F B s 10e4 34 363 0 349 ad B s 10e4 33 870 0 349 a 10 12 14 16 18 20 22 Cycle Tab 1 The table shows the average Ct values and the corresponding standard deviation of Bacillus subtilis spiked samples derived by real time PCR Fig 1 Real time PCR results from a representative RT PCR run with Bacillus subtilis samples performed in dilutions 10 purple 10 blue 10 dark green 10 light green 10 cyan 10 grey For testing of viral DNA and RNA 200 ul plasma was spiked with 2 ul hCMV or 2 ul Influenza stock solution respectively The detection was done by an in house real time PCR for hCMV and Influenza respectively All real time PCR s were performed on a Corbett Rotor Gene 3000 or Step One Plus Cycler Applied Biosystems 12 InviMag Universal Kit KF96 0515 c RNA Virus Detection nfluenza Norm Fluoro 10 1 Threshold 10 2 3 TR ST EE S H Gg Fig 2 Real time PCR results from a representative RT PCR run with nfluenza m samples performed in dilutions undiluted green 107 blue 10 orange PTC red NTC black d DNA Virus Detection hCMV Harm Fluore i Fig 3 Real time PCR results from a representative RT PCR run with hCMV ei samples Es in dilutions undiluted green 107 blue 10 orange PTC red NTC black No Color NEME Type OT itera hed Some oe 2 E eens wants sanoe e287 o E merc uranas samne 2208 JM messie s
3. 23 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 20 InviMag Universal Kit KF96 0515 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 Resuspend the bacterial pellet in 200 ul distilled water or PBS Prepare the 2 ml Deep Well Plate refers as Lysis Plate with Lysozyme for the preparation of bacterial DNA Use 20 ul Lysozyme for an improved lysis Add the 20 ul Lysozyme directly to the cavities of the Lysis Plate before adding samples or other reagents Transfer the 200 ul sample now into a cavity of a 2 ml Deep Well Plate refers as Lysis Plate A Add 200 ul Lysis Buffer HLT 20 ul Carrier RNA and 20 ul Proteinase K or B Add 240 ul Master Mix as described at point Lysis Procedure page 17 to each sample lt is also possible to add the Lysozyme to the Master Mix Use the same volumes as Proteinase K shown in the table of Lysis procedures page 17 for preparation of the Master Mix Prefill the remaining plates with required buffers and appropriate volumes and continue with the loading of the system see Starting a Run page 23 21 InviMag Universal Kit KF96 0515 Protocol 7 S
4. 95040450 InviMag Universal Kit KF96 0515 Stratecee molecular STRATEC Molecular GmbH Robert Rossle Str 10 13125 Berlin Germany Phone 49 30 94 89 29 01 Fax 49 30 94 89 29 09 E mail info berlin stratec com www stratec com 1G7002 05 2015
5. KF96 to which they apply are listed below as follows Lysis Buffer HLT Proteinase K contains guanidine hydrochloride f 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 7 InviMag Universal Kit KF96 0515 Product characteristics of the InviMag Universal Kit KF96 preparation up to 200 ul cell free body fluids like serum plasma or liquor etc up to 200 ul rinsed liquid from swabs Depends on sample storage and source most of the eluted nucleic acid s about 60 min up to 200 ul blood EDTA Citrate stabilized but not heparin Quantitative RT PCR is recommended for determination of the viral RNA or DNA yield 1x 10 mammalian c
6. Step 1 Beginning of step Mixing heating End of step Washing Step 2 Beginning of step Mixing heating End of step 26 96 DW tip comb Tip Plate Lysis Plate Precollect Release beads Mixing time speed Heating temperature C Preheat Postmix Collect beads Lysis Plate Message Dispensing volume ul Name Volume ul Name Volume ul Lysis Plate Precollect Release time speed Mixing time speed Heating during mixing Postmix Collect count Collect time s Wash Plate 1 Precollect Release time speed Mixing time speed Heating during mixing Postmix Collect count Collect time s Wash Plate 2 Precollect Release time speed Mixing time speed Heating during mixing Postmix Collect count Collect time s No Yes 00 15 00 Medium 75 Yes No No Add Isopropanol SNAPs 250 Isopropanol 230 SNAP Solution 20 No 00 00 10 Fast 00 05 00 Medium No o ow AZ No 00 00 10 Fast 00 05 00 Fast No No 4 5 No 00 00 10 Fast 00 04 00 Fast No No 4 5 InviMag Universal Kit KF96 0515 p Washing Step 3 Beginning of step Mixing heating End of step Drying Step Elution Step Beginning of step Mixing heating End of step Bead Removal Step Leave 27 Wash Plate 3 Precollect Release time speed Mixing time speed Heating during mixing Postmix Collect count Collect time s Wash Plate 3
7. all remaining plates with the required buffers and appropriate volumes and continue with the loading of the system see Starting a Run page 23 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 a 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 For the preparation of bacterial DNA we recommend to use 20 ul Lysozyme for an improved lysis Add the 20 ul Lysozyme directly to the Lysis Plate before adding samples or other reagents or Master Mix Transfer than 200 ul of the rinse liquid into a cavity of a 2 ml Deep Well Plate refers as Lysis Plate A Add 200 ul Lysis Buffer HLT 20 ul Proteinase K and 20 ul Carrier RNA to each sample If genomic DNA shall be prepared the addition of Carrier RNA is optional or B Add 240 ul Master Mix as described at point Lysis Procedure page 17 to each sample If genomic DNA shall be prepared the addition of Carrier RNA to the Master Mix is optional For bacterial DNA don t forget to add the Lysozyme at first in the plate Prefill all remaining plates with the required buffers and appropriate volumes and continue with the loading of the system see Starting a Run page 23 b the sample will not be used for cultivation Cut off the relevant part of the
8. and 20 pl SNAP Solution magnetic beads Internal extraction controls should be added during this pause step too 2 Binding of the nucleic acids Binding step for 5 min SNAP separation Transportation of the SNAP bound nucleic acids into Washing Plate 1 3 First Washing Step Sample washing for 5 min SNAP separation Transportation of the SNAP bound nucleic acids into the Washing Plate 2 4 Second Washing Step Sample washing for 4 min SNAP separation Transportation of the SNAP bound nucleic acids into the Washing Plate 3 5 Third Washing and Drying Step Sample washing for 3 min SNAP separation Air drying of the SNAP bound nucleic acids outside the plate for 5 min Transportation of the SNAP into the Elution Plate 6 Elution of the nucleic acids Incubation of the SNAP bound nucleic acids into the Elution Plate 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 Washing Plate_3 disposal Important Notes After finishing the extraction protocol the Elution Plate 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 and centrifuge at max speed for
9. broad panel of downstream applications like PCR real time PCR Restriction Enzyme Digestion HLA Typing o Southern Blot O OO 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 robotic station 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 a centrifuge vacuum manifold or on other robotic stations as well as magnetic bead based kits See page 23 The PCR process is covered by US Patents 4 683 195 and 4 683 202 and foreign equivalents owned by Hoffmann La Roche AG 8 InviMag Universal Kit KF96 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 Cultivated bacteria or bacterial suspension s Bacteria have to be pelleted after cultivation and resuspended in defined conditions Best results are obtained with fresh material Swabs Saliva The protocol works with fresh saliva prepared swabs as well as with dried swabs The protocol is not validated for the isolation of DNA from swabs provided in storage buffers from other providers Best results are obtained using freshly
10. extracted samples 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 However viral RNA purification should be processed as soon as possible Urine The bacteria should be pelleted while the supernatant is discarded urea contaminations can inhibit PCR reactions For some applications fresh urine can be used directly Best results are obtained with freshly pelleted material 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 30 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 quickly realize 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 derived from blood
11. research use only 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 1 InviMag Universal Kit KF96 0515 Table of content Kit contents of InviMag Universal Kit KF96 3 Kit contents of InviMag Universal Kit KF96 w o plastic 4 Symbols 5 Storage 5 Quality Control and product warranty 5 Intended use 6 Product use limitation 6 Safety information 7 Product characteristics of the InviMag Universal Kit KF96 8 Sampling and storage of starting material 9 Principle and procedure 10 Procedure 10 Yield and quality of genomic DNA derived from blood 10 Yield and quality of viral nucleic acids 11 Protocol validation 11 Important notes 14 Preparing reagents and buffers 14 Reagents and equipment to be supplied by user 15 Important indications 15 Scheme of the InviMag Universal Kit KF96 16 Lysis procedures 17 Protocol 1 Simultaneous isolation of nucleic acids viral DNA
12. 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 rinse liquid from swabs or bacterial suspensions sputum BAL using magnetic beads and the KF96 KFflex96 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 the plates allowing safe handling of potentially infectious samples The isolation protocols and buffers are optimized to provide high yields and purities However for reproducible yields 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 INTENDED FOR USE BY PROFESSIONALS 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 diagnostic assay should be interpreted with regards to other clinical or laboratory findings All utilities except Ethanol and l
13. swab and transfer this part into a 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 we recommend to use 20 ul Lysozyme for an improved lysis Add the Lysozyme directly to the Lysis Plate before adding samples or other reagents Transfer than 200 ul of the rinsed liquid into a cavity of a 2 ml Deep Well Plate refers as Lysis Plate 18 InviMag Universal Kit KF96 0515 A Add 200 ul Lysis Buffer HLT 20 ul Proteinase K and 20 ul Carrier RNA to each sample If genomic DNA shall be prepared the addition of Carrier RNA is optional Or B Add 240 ul Master Mix as described at point Lysis Procedure page 17 to each sample If genomic DNA shall be prepared the addition of Carrier RNA to the Master Mix is optional For bacterial DNA don t forget to add the Lysozyme at first in the plate see above Prefill all remaining plates with the required buffers and appropriate volumes and continue with the loading of the system see Starting a Run page 23 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 For the preparation of bacterial DNA we recommend to use 20 ul Lysozyme for an improved lysis Add the Lysozyme directly to the Lysis Plate before adding samples or other reage
14. the Lysozyme at first in the plate see above Prefill the remaining plates with required buffers and appropriate volumes and continue with the loading of the system see Starting a Run page 23 19 InviMag Universal Kit KF96 0515 Protocol 5 Simultaneous isolation of total nucleic acids from sputum Please read the protocols carefully prior to the start of the preparation procedure Transfer 150 ul from the soutum sample into a 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 For viral NA preparation the temperature is not disturbing the next steps Prepare the 2 ml Deep Well Plate refers as Lysis Plate with Lysozyme for the preparation of bacterial DNA During this time the temperature of the sample will decrease and prevent an inactivation of Lysozyme Use 20 ul Lysozyme for an improved lysis Add the 20 ul Lysozyme directly to the cavities of the Lysis Plate before adding samples or other reagents Transfer 200 ul from the sample mixture into the cavity of a 2 ml Deep Well Plate refers as Lysis Plate A Add 200 ul Lysis Buffer HLT 20 ul Carrier RNA and 20 ul Proteinase K If only genomic DNA is processed the addition of Carrier RNA is optional or B Add 240 ul Master Mix as described at point Lysis Procedure page 17
15. to each sample Prefill the remaining plates with required buffers and appropriate volumes and continue with the loading of the system see Starting a Run page 23 Protocol 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 Non viscous samples Transfer 1 ml of tracheal secret or BAL into a 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 a cavity of a 2 ml Deep Well Plate refers as Lysis Plate A Add 200 ul Lysis Buffer HLT 20 ul Carrier RNA and 20 ul Proteinase K For the preparation of bacterial DNA we recommend to use 20 ul Lysozyme for an improved lysis Add the 20 ul Lysozyme each cavity directly into the Lysis Plate before adding samples or other reagents Or B Add 240 ul Master Mix as described at point Lysis Procedure page 16 to each sample For the preparation of bacterial DNA we recommend to use Lysozyme for an improved lysis Use the same volumes as Proteinase K shown in the table of Lysis procedures page 17 for preparation of the Master Mix Prefill the remaining plates with required buffers and appropriate volumes and continue with the loading of the system see Starting a Run page
16. 1 min Then transfer the nucleic acids containing supernatant into the provided Receiver Tubes The eluted nucleic acids are ready to use in different downstream applications 24 InviMag Universal Kit KF96 0515 For self programming of the KF96 and KFflex96 instrument Keagent info Tip Plate Name Lysis Plate Name Samp k Lysi Buffer HLT Proteinase E Camiea ERA Lysozyme required Wash Plate 1 Wash Butter HLT Wash Plate 7 Name Wash Buffer M Wash Plate 3 Wash Butter I Elution Plate Name Elution Boffer M Dispensed reagents Lysis Flate Name Isopropanol SNAP Solution Well volume w Well volume yl ba tal fa 3 3 Step Adjust Binding Cond ition Adjust Binding Co md ition 29 King Fisher 96 KF plate Total reagent volume ul Microtiter DW 96 plate Total reagent volume HI Afr otiter DW 96 plate Total reagent volume m Aic otter DW 96 pate Total reagent volume ul Aic otter DW 96 pate Total reag ent volume ul Kine Fisher 96 KF plate Total reagent volume m Mficrotiter DW 96 plate Well volume ul 230 r 30 Samp kb Reagent Reagent Reagent Faagant Total reagent volume ul InviMag Universal Kit KF96 0515 Steps data Int Tip 1 p Pick Up Lysis Step Beginning of step Mixing heating End of step Adjust Binding Condition Reagent s Binding Step Beginning of step Mixing heating End of step Washing
17. 2 preps 5 x 96 preparations 75 preparations 250 preparations 250 preparations 250 preparations 5 x 96 preparations 5 x 96 preparations 75 preparations 250 preparations 75 preparations Possible suppliers for lsopropanol Carl Roth 2 Propanol Applichem Sigma Rotipuran gt 99 7 p a ACS ISO 2 Propanol 2 Propanol Order No 6752 Possible suppliers for centrifuges Eppendorf AG 22331 Hamburg Germany Phone 49 0 40 53801 0 Fax 49 0 40 53801 556 E Mail eppendorf eppendorf com Internet www eppendorf com KingFisher 96 and consumables KingFisher 96 Magnetic Particle Processor 100 240V 50 60Hz KingFisher 96 Head for Deep Well plate KingFisher 96 tip comb for PCR magnets 8 x 10 pcs box KingFisher 96 tip comb for KF magnets 10 x 10 pcs box KingFisher 96 tip comb for DW magnets 10 x 10 pcs box KingFisher 96 KF plate 200ul 48 plates box Microtiter deep well 96 plate 50 plates box Order No A3928 SIGMA Laborzentrifugen GmbH 37507 Osterode am Harz Germany Phone 49 5522 5007 0 Fax 49 5522 5007 12 E Mail info sigma zentrifugen de Internet www siama zentrifuagen de 33 Catalogue No 7450300100 7450300200 7450300150 7450300250 Catalogue No 7450330300 7450330400 2450120100 7450300200 2450110300 1040500300 1040100300 1033200300 7443300200 7442300200 2441150200 1440100300 2433150200 Order No 59304 1L F 9400500 24073430 97002514 97002524 97002534 97002540
18. 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 Oe This kit should only be used by trained personnel 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 disturbed by the included Carrier RNA as well as DNA or RNA which is co purified 32 InviMag Universal Kit KF96 0515 Ordering information Product InviMag Universal Kit KF96 InviMag Universal Kit KF96 InviMag Universal Kit KF96 w o plastic InviMag Universal Kit KF96 w o plastic Related Products InviMag Universal Kit STARIet InviMag Universal Kit STARIet InviMag Universal Kit IG InviMag Pathogen Kit KF96 InviMag Pathogen Kit KFmL RTP Pathogen Kit RTP DNA RNA Virus Mini Kit RTP Bacteria DNA Mini Kit InviMag Virus RNA Mini Kit KF 96 InviMag Virus DNA Mini Kit KF 96 InviMag Virus DNA RNA Mini Kit KEmL InviMag Virus DNA RNA Mini Kit InviMag Bacteria DNA Mini Kit KFmL Package Size 1 x 96 preparations 5 x 96 preparations 1 x 96 preparations 5 x 96 preparations Package Size 4 x 96 preparations 24 x 96 preparations 8 x 1
19. Kit KF96 0515 Principle and procedure The InviMag Universal Kit KF96 procedure comprises following steps Sample preparation if required Lysis step 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 G OD Procedure Bacteria must be cultivated at special conditions An aliquot of the bacteria suspension is used to achieve a bacterial pellet by centrifugation at high speed for 5 min while the supernatant is discarded Pretreatment Please check the specific protocol section 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 lf required add the extraction controls 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 while the nucleic acids remain bound to the magnetic beads Eluti
20. 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 30 InviMag Universal Kit KF96 0515 General notes on handling DNA Nature of DNA The length and delicate physical nature of DNA requires careful handling to avoid damage due to shearing and 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 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 nitro
21. RNA from cell free body fluids or blood genomic DNA 17 Protocol 2 Simultaneous isolation of nucleic acids DNA RNA from swab material 18 Protocol 3 Simultaneous isolation of nucleic acids DNA and RNA from tissue biopsies 19 Protocol 4 Isolation of DNA from bacteria pellets up to 1 x 10 bacterial cells 19 Protocol 5 Simultaneous isolation of total nucleic acids from sputum 20 Protocol 6 Simultaneous isolation of total nucleic acids DNA and RNA from tracheal secretes or BAL 20 Protocol 7 Simultaneous isolation of viral nucleic acids from stool samples 22 Protocol 8 solation of bacterial DNA from stool samples 22 Starting a Run 23 For self programming of the KF96 and KFflex96 instrument 25 Troubleshooting 28 Appendix 30 General notes on handling DNA 31 General notes on handling RNA 32 Ordering information 33 2 InviMag Universal Kit KF96 0515 Kit contents of InviMag Universal Kit KF96 Store the MAP Solution B 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 Component Catalogue No Lysozyme Buffer Lysozyme Lysis Buffer HLT Carrier RNA RNAse Free Water Proteinase K SNAP Solution Binding Solution fill with 99 7 Isopropanol Wash Buffer HLT Wash Buffer II Wash Buffer M Elution Buffer M KF96 Tip Comb for DW magnets 2 0 ml Deep Well Plate 200 ul Elution Plate Sealing Foils 1 5 ml Receiver Tubes Manu
22. Sstratecee molecular 9 User manual InviMag Universal Kit KF96 for use on KingFisher 96 and KingFisher Flex Thermo Fisher Scientific for automated purification of DNA genomic bacterial mitochondrial and viral as well as viralRNA from up to 200 ul of clinical samples with magnetic beads 7A50300X0 anal STRATEC Molecular GmbH D 13125 Berlin d Instruction for InviMag Universal Kit KF96 The InviMag Universal Kit KF96 combines the advantages of the innovative InviMag technology with easy handling of magnetic particles in combination with either the KF96 or KFflex96 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 of total genomic 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 KF96 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
23. al or B Add 240 ul Master Mix as described at point Lysis Procedure page 17 to each sample If genomic DNA shall be prepared the addition of Carrier RNA to the Master Mix is optional Prefill the remaining plates with required buffers and appropriate volumes and continue with the loading of the system see Starting a Run page 23 17 InviMag Universal Kit KF96 0515 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 A Add 200 ul Lysis Buffer HLT 20 ul Proteinase K and 20 ul Carrier RNA into each cavity of a 2 ml Deep Well Plate refers as Lysis Plate If genomic DNA shall be prepared the addition of Carrier RNA is optional For the preparation of bacterial DNA we recommend to use 20 ul Lysozyme for an improved lysis Add the Lysozyme directly to the Lysis Plate before adding samples or other reagents or B Add 240 ul Master Mix as described at point Lysis Procedure page 17 to into each cavity of a 2 ml Deep Well Plate refers as Lysis Plate lf genomic DNA shall be prepared the addition of Carrier RNA to the Master Mix is optional Insert the swab into the cavity of the Lysis Plate Incubate for 5 10 min at RI and stir occasionally After incubation remove the swab and squeeze it out inside the cavity to remove residual liquid and then discard the swab Prefill
24. al Initial steps 1 x 96 preparations 7450300100 15 ml 150 mg 30 ml 2 x 1 2 ml working solution 3X2 ml 2 x 1 1 ml working solution 2x1 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 DRA 2 2 x 50 pcs 1 Add 60 ml of abs 99 7 Isopropanol to the bottle Wash Buffer HLT mix thoroughly and keep the bottle firmly closed Add the provided amount of Lysozyme to the bottle with the Lysozyme Buffer and mix it thoroughly 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 each tube Carrier RNA in 1 2 ml RNAse free water Mix thoroughly until completely dissolving Resuspend each tube Proteinase K in 1 1 ml RNAse free water mix thoroughly until completely dissolving and store at 20 C Fill 30 ml 99 7 Isopropanol molecular biologic grade into the empty bottle 5 X 96 preparations 7450300200 15 ml 150 mg 120 ml 15 ml working solution 2x15ml 10 5 ml working solution 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 5 5x4 5x2 10 10 x 50 pcs 1 Add 240 ml of abs 99 7 Isopropanol to the bottle Wash Buffer HLT mix thoroughl
25. defects shall be reported to STRATEC Molecular immediately upon detection thereof The chemicals and the plastic parts are for laboratory use only They must be stored in the laboratory and must not be used for other purposes than intended The included chemicals are only useable once and are not suitable for consumption 6 InviMag Universal Kit KF96 0515 Safety information When and while working with chemicals always wear a suitable lab coat disposable gloves and protective goggles Avoid skin contact with reagents and samples 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 KF96 procedures for residual infectious materials Contamination of the liquid waste with residual infectious materials is highly unlikely but cannot be excluded completely Therefore liquid waste must be considered and handled as infectious and discarded accordingly to local safety regulations European Community risk and safety phrases for the components of the InviMag Universal Kit
26. e 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 guarantees the correct function of the InviMag Universal Kit KF96 for applications as described in the manual In accordance with STRATEC Molecular s certified QM System each component of the InviMag Universal Kit KF96 was tested against predetermined specifications to ensure consistent product quality All products sold by STRATEC Molecular are subjected to extensive quality control procedures according to ISO 9001 2008 and ISO 13485 2003 AC 2009 and are warranted to perform as described when used correctly Any problems should be reported immediately STRATEC Molecular reserves the right to change alter or modify any product to enhance its performance and design at any time If you have any questions or problems regarding any aspects of InviMag Universal Kit KF96 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 2907 2903 or contact your local distributor 5 InviMag Universal Kit KF96 0515 Intended use The InviMag Universal Kit KF96 is designed for semi automated rapid and economical preparation of nucleic acids viral DNA RNA genomic DNA bacterial DNA but not plasmid DNA from 200 ul
27. e reaction Eluates derived by this kit will contain Carrier RNA which will greatly exceed the amount of the isolated 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 according 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 Protocol validation The provided extraction protocols were intensively tested on a KingFisher Flex 96 instrument with the provided reagents and consumables Typical results for the extraction of bacterial DNA genomic DNA derived from blood viral DNA and RNA are shown below Actual results can vary depending upon sample age quality type and the species used Samples For testing the isolation efficiency of bacterial DNA frozen bacterial pellets from the gram positive bacterium Bacillus subtilis were used in dilutions from 1 x 10
28. einase K and 20 ul Carrier RNA optional for genomic DNA For a bacterial DNA preparation 20 ul of Lysozyme should be added in the first step Continue with the respective lysis protocol Prefill all plates with required buffers and appropriate volumes Tip Plate Lysis Plate Washing Plate_1 Washing Plate 2 Washing Plate 3 Elution Plate Insert the KF96 Tip Comb for DW magnets on a Tip Plate See lysis procedures page 17 for respective protocol Add 900 ul Wash Buffer HLT to a 2 0 ml Deep Well Plate Add 900 ul Wash Buffer M to a 2 0 ml Deep Well Plate Add 1000 ul Wash Buffer II to a 2 0 ml Deep Well Plate Add 100 ul Elution Buffer M to the Elution Plate same size as Tip Plate Please read the protocols carefully prior to the start of the preparation procedure The following steps are performed on the KingFisher instrument Lysis of the sample After lysis during the pause step please add 230 ul Binding Solution and 20 ul SNAP Solution 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 Elution Plates and Tip Plates are identically Use one provided Elution Plate as a Tip Plate 16 InviMag Universal Kit KF96 0515 Lysis procedures For easier handling we rec
29. ells 10 mg tissue sample The InviMag Universal Kit KF96 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 King Fisher instrument uses magnetic rods to transport the DNA RNA binding magnetic particles through the various extraction phases lysis binding washing and elution The automated purification process results in a fast reliable and robust technique After a sample specific lysis using Lysis Buffer HLT and Proteinase K and Lysozyme if required optimal binding conditions are adjusted upon addition of Binding Solution The genomic DNA and or viral DNA RNA will bind to the added magnetic particles SNAP Solution and is separated from the solution by magnetic rods controlled by the KingFisher system Subsequent to three washing steps of the particle bound nucleic acids the nucleic acids are finally eluted in Elution Buffer M Due to the high purity the eluted nucleic acids are ready to use in a
30. emne ear BI IN NEE fesa BI IN TEE as 7E merear Sage ar DI IE NEE DI IN EE EE DI EEN IE El INEEN Dl INEEN IM we beseer Tab 2 The table shows the estimated Ct values of Influenza spiked samples performed in dilutions and derived by real time PCR No Color NEME Type OT powa Some f e IM vw ates sarme f Be NE Mr O hon ie sege fre IM hon ie sege pror IM ron ie samme perar EI INEEN DI rowo sanoe foose DI IE NEE Dl IERE EED IM Pre Posne cona aus eB wre ewe conr ol wre ese conrra Tab 3 The table shows the estimated Ct values of hCMV spiked samples performed in dilutions and derived bv real time PCR InviMag Universal Kit KF96 0515 Important notes Immediately upon receipt of the product inspect the product and its components as well as the package for any apparent damages correct quantities and quality If there are any unconformities immediately notify STRATEC Molecular in writing with immediate effect upon inspection thereof If buffer bottles are damaged contact the STRATEC Molecular Technical Services or your local distributor In case of liquid spillage refer to Safety Information see page 7 Do not use damaged kit components 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 w
31. g 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 29 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 Mix SNAP Solution thoroughly before pipetting to the Deep Well Plate Elute the RNA with lower volume of Elution Buffer R Change the modified volume in the run file too Ensure that the storage of starting material was correctly avoid repeated thawing and freezing cycles of the sample material Ensure that the correct amount of ethanol isopropanol is added to the Wash Buffers ensure that the storage of starting material was correctly Avoid multiple thawing and freezing of the sample material Ensure that the starting material is fresh or stored at appropriate conditions 20 C 80 C Avoid multiple thawing and freezing of the material increase drying time for removing 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 tem
32. gen 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 assay 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 31 InviMag Universal Kit KF96 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 mater
33. he 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 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 5 ml RNAse free water mix thoroughly until completely dissolving and store at 20 C Fill 120 ml 99 7 Isopropanol molecular biologic grade into the empty bottle see order information 4 pcs 1 pcs 2 pcs 5 x 4 pcs 5 PCs 5 X 2 pcs InviMag Universal Kit KF96 0515 Symbols Manufacturer Lot number Catalogue number Expiry date Consult operating instructions g T Erag E EE ha Xt Temperature limitation Q Do not reuse Storage All buffers and kit contents of the InviMag Universal Kit KF96 except Carrier RNA dissolved Proteinase K 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 should be stored at 2 8 C Dissolved Carrier RNA and Proteinase K must be stored at 20 C in aliquots 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 th
34. he Proteinase K in 10 5 ml RNAse free water mix thoroughly until completely dissolving and store at 20 C Add 120 ml 99 7 Isopropanol molecular biologic grade to the empty bottle labeled with Binding Solution 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 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 OD The InviMag Universal Kit KF96 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 2 Propanol Applichem Sigma Rotipuran gt 99 7 p a ACS ISO 2 Propanol f r die Molekularbiologie 2 Propanol Order no 6752 Order no A3928 Order no 59304 1L F 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 Elution Buffer M with DNases RNases Storing samples Frozen serum or plasma samples should not be thawed more than once Repeated freezing and thawing cyc
35. ial 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 both inactivate RNAses and ensure that no other nucleic acids such as plasmid DNA are present on the surface of the glassware You can also 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 ethanol 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 You can also use chloroform resistant plastic ware rinsed with chloroform to inactivate RNAses All solutions must be prepared with DEPC treated RNAse free water
36. imultaneous isolation of viral nucleic acids from stool samples Please read the protocols carefully prior to the start of the preparation procedure Transfer 100 ul 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 virus containing supernatant into the cavity of a2 ml Deep Well Plate refers as Lysis Plate A Add 200 ul Lysis Buffer HLT 20 ul Carrier RNA and 20 ul Proteinase K or B Add 240 ul Master Mix as described at point Lysis Procedure page 17 to each sample Prefill the remaining plates with required buffers and appropriate volumes and continue with the loading of the system see Starting a Run page 23 Protocol 8 Isolation of bacterial DNA from stool samples Please read the protocols carefully prior to the start of the preparation procedure 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 Prepare the 2 ml Deep Well Plate refers as Lysis Plate with Lysozyme for the preparation of bacterial DNA Use 20 ul Lysozyme for an improved lysis Add the 20 ul Lysozyme directly to the cavities of the Lysis Plate before adding samples or other reagents Transfer 200 ul of the bacteria containing supernatant into the cavity of a 2 ml Deep Well Plate refers a
37. l Wash Buffer Il into the cavities of a Deep Well Plate Elution Plate Add 100 ul Elution Buffer M into the cavities of a Elution Plate 3 Choose the assay file InviMag Universal KF96 or InviMag_Universal_ KFflex96 on the display of the instrument depends on the used instrument and press the START button 4 Insert the prefilled plates onto the right position of the KingFisher surface by following the specification shown on the display Confirm every step by pressing the START button 5 If all prefilled plates are loaded into the system press the START button again to start the assay file 6 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 the Lysis Pate 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 15 7 Reinsert the 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 23 InviMag Universal Kit KF96 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
38. les may lead to denaturation and precipitation of proteins resulting in reduced titers and therefore reduced yields 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 present 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 denaturated completely by the Lysis Buffer Internal Controls The use of an internal control is recommended when using the InviMag Universal Kit KF96 in combination with diagnostic amplification systems Internal controls should be added directly to the lysis mixture during the pause step where the Binding Solution and beads are added Do not add any controls to the sample or to the lysis mixture until the lysis step is complete In rare cases the controls may be degraded by 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 volume of 1 2 ml Carrier RNA tube 15 InviMag Universal Kit KF96 0515 Scheme of the InviMag Universal Kit KF96 Please read protocols prior the start of the preparation carefully Transfer 200 ul Lysis Buffer HLT and 200 ul sample into a cavity of a 2 ml Deep Well Plate refers as Lysis Plate Add 20 ul Prot
39. lution Buffer R 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 isopropanol is added to the Wash Buffers and stored correctly 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 thawing and freezing cycles of the material Due to the very gentle isolation procedure it may happen that isolated genomic DNA forms a ball To overcome this the 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 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 to a new tube InviMag Universal Kit KF96 0515 Probable cause Comments and suggestions Low amount 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 startin
40. n 2x1 1ml 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 2 2 X 50 pcs J Add 60 ml of abs 99 7 Isopropanol to the bottle Wash Buffer HLT mix thoroughly and keep the bottle firmly closed Add the provided amount of Lysozyme to the bottle with the Lysozyme Buffer and mix it thoroughly 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 each tube Carrier RNA in 1 2 ml RNAse free water Mix thoroughly until completely dissolving Resuspend each tube Proteinase K in 1 1 ml RNAse free water mix thoroughly until completely dissolving and store at 20 C Fill 30 ml 99 7 Isopropanol molecular biologic grade into the empty bottle 5 x 96 preparations 7450300250 15 ml 150 mg 120 ml 15 ml working solution 2x15ml 10 5 ml working solution 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 10 10 x 50 pcs J Add 240 ml of abs 99 7 Isopropanol to the bottle Wash Buffer HLT mix thoroughly and keep the bottle firmly closed Add the provided amount of Lysozyme to the bottle with the Lysozyme Buffer and mix it thoroughly Add 450 ml of 96 100 ethanol to t
41. nts Transfer 1 10 mg from the tissue biopsy sample into a cavity of a 2 ml Deep Well Plate refers as Lysis Plate and add 200 ul distilled water 200 ul Lysis Buffer HLT 20 ul Carrier RNA and 20 ul Proteinase K to each sample or add 240 ul Master Mix as described at point Lysis Procedure page 16 to each sample If genomic DNA shall be prepared the addition of Carrier RNA to the Master Mix or to the sample is optional Prefill the remaining plates with required buffers and appropriate volumes and continue with the loading of the system see Starting a Run page 23 Protocol 4 Isolation of DNA from bacteria pellets up to 1x 10 bacterial cells or from non viscous tracheal secrete of BAL Please read the protocols carefully prior to the start of the preparation procedure 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 For an improved lysis add 20 ul Lysozyme directly to the Lysis Plate before adding samples or other reagents Resuspend the bacterial pellet in 200 ul distilled water or PBS and transfer the sample into a cavity of a 2 ml Deep Well Plate refers as Lysis Plate A Add 200 ul Lysis Buffer HLT 20 ul Carrier RNA and 20 ul Proteinase to each cavity or B Add 240 ul Master Mix as described at point Lysis Procedure page 17 to each sample Dont forget to add
42. ommend 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 use a volume of 5 greater than that required Attention Please be aware that you have to prepare the Master Mix shortly before carrying out the purifications adapted to the number of samples that will be processed Longer incubation will decrease the activity of the Proteinase K Preparation of a Master mix esl Ha oi Amount of Lysis Buffer HLT Amount of Carrier RNA Amount of Proteinase K ji tisammle _ _2041samples _ _20 serps _ 1 7 ml 170 ul oa som O soom O soom 8 mt cow O 40 84m hot ho OO ag 100m ft pt 6 00 pI O 11 8 ml 1180 pl 80 16 8 mI 1680 UJ 88 18 5 ml 1850 ul 20 2 ml 2020 ul 2020 ul Protocol 1 Simultaneous isolation of total nucleic acids 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 fill up to a total volume of 200 ul with ddH2O or PBS Transfer 200 ul sample into a cavity of a 2 ml Deep Well Plate refers as Lysis Plate A Add 200 ul Lysis Buffer HLT 20 ul Proteinase K and 20 ul Carrier RNA to each sample If genomic DNA shall be prepared the addition of Carrier RNA is option
43. on The nucleic acids are finally eluted in Elution Buffer M and are ready to use in different subsequent downstream applications e g for 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 KF96 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 3 x 10 to 1 x 10 cells ml will yield 3 6 ug of genomic DNA If a whole blood sample is mixed with anticoagulant containing buffer solutions the overall leucocyte concentration decreases and the yield is reduced Please keep in mind that added Carrier RNA will falsify the real genomic DNA content in photometric measurements 10 InviMag Universal Kit KF96 0515 Yield and quality of viral nucleic acids The amount of purified nucleic acids in the InviMag Universal Kit KF96 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 pathogen detection system The tests should be performed accordingly to manufacturers specifications Different amplification systems vary in efficiency depending on the total amount of nucleic acid present in th
44. orking 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 Preparing reagents and buffers Prior to each isolation Before starting a run bring all reagents to room temperature Where necessary gently mix and re dissolve any precipitates by warming to 30 C until dissolved Swirl gently to avoid foaming Proteinase K Add described amount of ddH2O see table below to needed tube or bottle of Proteinase K mix thoroughly and store not needed Proteinase K at 20 C Dividing the Proteinase K into aliquots to avoid repeated freezing and thawing is recommended Lysozyme Lysozyme should be prepared directly before starting the isolation Solve the lyophilized Lysozyme within the bottle of Lysozyme Buffer Mix thoroughly until all of the Lysozyme is solved Store not directly used Lysozyme solution in aliquots at 20 C and avoid repeated freezing and thawing Wash Buffer and Wash Buffer Il Before use add the described volume of 96 100 ethanol to the bottle with Wash Buffer and II as described below After adding
45. perature Centrifuge at full soeed for 1 min and transfer supernatant into a new tube InviMag Universal Kit KF96 0515 Appendix KingFisher Bindlt Software 3 2 or higher versions Bindlt software 3 2 or higher versions were and may be used to create assay files for the KFmL KF96 KFflex96 or KF Duo instruments The provided assay file s can either be transferred onto the corresponding workstation s or be started directly from within the Bindlt software after assay import Please keep in mind that assay s run from within the Bindlt software are not stored in the workstation memory Important Be advised that Bindlt SW 3 2 or higher versions use a new unique file extension Therefore it is not possible to import assay files created with Bindlt 3 2 or higher versions into older Binalt software versions Please ask your local Thermo Scientific distributor for a software update Note When creating assay files for usage with KingFisher instruments in combination with Microtiter Deep Well plates e g Thermo Electron it is essential to use the KingFisher software 3 2 or higher versions for assay development because this software version includes the correct adjustments for the microtiter plate It is highly recommended to use Thermo Microtiter Deep Well plates with KF96 KFflex96 KF Duo workstations to ensure the best purification result Minimum system requirements for Bindlt Software 3 2 or higher versions PC requirements Supported
46. s Lysis Plate A Add 200 ul Lysis Buffer HLT 20 ul Carrier RNA and 20 ul Proteinase K or B Add 240 ul Master Mix as described at point Lysis Procedure page 17 to each sample lt is also possible to add the Lysozyme to the Master Mix Use the same volumes as Proteinase K shown in the table of Lysis procedures page 17 for preparation of the Master Mix Prefill the remaining plates with required buffers and appropriate volumes and continue with the loading of the system see Starting a Run page 23 22 InviMag Universal Kit KF96 0515 Starting a Run I Preliminary Steps to process the sample onto the KingFisher System Important For working with the KF96 KFflex96 instrument please carefully read the manufacturer s manual before using the system for the first time 1 Switch on the KF96 KFflex96 instrument Tip Plate Place the KF96 Tip Comb for DW magnets on an Elution Plate Tip Plate Note Use one provided Elution Plate as Tip Plate These are identical 2 Prefill the all Deep Well Plates with the required buffers and appropriate volumes Important Mix the bottle with the SNAP Solution by vigorously vortexing before usage Lysis Plate see the corresponding isolation protocol chapter Lysis procedures page 17 Washing plate_1 Add 900 ul Wash Buffer HLT into the cavities of a Deep Well Plate Washing plate_2 Add 900 ul Wash Buffer M into the cavities of a Deep Well Plate Washing plate 3 Add 1000 u
47. sopropanol required for preparation of nucleic acids are provided by the InviMag Universal Kit KF96 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 a 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 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 quality control procedures according to ISO 9001 2000 and ISO EN 13485 and are warranted to perform as described herein Any problems incidents or
48. the ethanol mix shortly and keep the bottles always firmly closed Wash Buffer HLT Before use add the described volume Isopropanol to the bottle with Wash Buffer HLT as described below After adding mix shortly and keep the bottles always firmly closed 1 x 96 extractions Add 60 ml of abs 99 7 Isopropanol to the bottle Wash Buffer HLT and mix thoroughly Add 90 ml of 96 100 ethanol to the bottle Wash Buffer M and mix thoroughly Add 105 ml of 96 100 ethanol to the bottle Wash Buffer II and mix thoroughly Add the provided amount of Lysozyme to the bottle with the Lysozyme Buffer and mix it thoroughly Resuspend each tube Carrier RNA in 1 2 ml RNAse free water Mix thoroughly until completely dissolving Resuspend each tube Proteinase K in 1 1 ml RNAse free water mix thoroughly until completely dissolving and store at 20 C Add 30 ml 99 7 Isopropanol molecular biologic grade to the empty bottle labeled with Binding Solution 14 InviMag Universal Kit KF96 0515 5 x 96 extractions Add 240 ml of abs 99 7 Isopropanol to the bottle Wash Buffer HLT and mix thoroughly Add 450 ml of 96 100 ethanol to the bottle Wash Buffer M and mix thoroughly Add 420 ml of 96 100 ethanol to the bottle Wash Buffer Il and mix thoroughly Add the provided amount of Lysozyme to the bottle with the Lysozyme Buffer and mix it thoroughly Resuspend the Carrier RNA in 15 ml RNAse free water Mix thoroughly until completely dissolving Resuspend t
49. to 1 x 10 The bacteria were grown in an over night culture and the derived cell pellets from 1 ml of this culture were stored at 20 C until further use In all experiments a fresh pellet was used from the 20 C stock The detection was done by an in house Bacillus subtilis real time PCR based detection assay performed on a Step One Plus Cycler Applied Biosystems For the extraction process 200 ul of the corresponding dilution was used respectively For testing of genomic DNA derived from whole blood 200 ul deeply frozen mammalian blood was used The blood samples were either stabilized with EDTA or citrate a Genomic DNA human whole blood Fig 4 Shown is a representative gel picture of genomic DNA 10 ul eluate lane derived from human whole blood N EA add W d separated on a 0 8 agarose gel The staining was performed with ethidium bromide The average purity OD260 OD320 calculated by photometric measurement is usually in the range of 1 8 2 1 An average yield of about 3 4 ug DNA is derived from 200 ul human whole blood 11 InviMag Universal Kit KF96 0515 b Bacterial Detection Bacillus subtilis Amplification Plot pea Sample Cr Cr SD perd B s 10e9 16 545 0 199 37 500 B s 10e9 16 826 0 199 od B s 10e8 19 740 0 163 seen B s 10e8 19 971 0 163 se B s 10e7 23 201 0 112 20 000 B s 10e7 23 043 0 112 yee B s 10e6 26 334 0 348 Ee B s 10e6 26 826 0 348 Ser B s 10e5 32 639 1 295 2500 B s 10e5 30 808
50. treated with anticoagulants like EDTA or Citrate but not with heparin synovial fluid samples or other cell free body fluids and rinse liquids from swabs can be used for extraction For short term storage samples can be kept on ice for 1 2 hours For up to 24 h 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 In addition cryoprecipitates formed during freeze thawing cycles can cause problems If cryoprecipitate are visible centrifuge them down 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 winning of the cell culture supernatant Repeated freezing and thawing of stored samples can negatively influence the sensitivity STRATEC Molecular will be released of its responsibilities if other sample materials than described in the Intended Use are processed or if the sample preparation protocols are changed or modified 9 InviMag Universal
51. y and keep the bottle firmly closed Add the provided amount of Lysozyme to the bottle with the Lysozyme Buffer and mix it thoroughly 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 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 5 ml RNAse free water mix thoroughly until completely dissolving and store at 20 C Fill 120 ml 99 7 Isopropanol molecular biologic grade into the empty bottle InviMag Universal Kit KF96 0515 Kit contents of InviMag Universal Kit KF96 w o plastic Store the MAP Solution B 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 Component Catalogue No Lysozyme Buffer Lysozyme Lysis Buffer HLT Carrier RNA RNAse Free Water Proteinase K SNAP Solution Binding Solution fill with 99 7 Isopropanol Wash Buffer HLT Wash Buffer Il Wash Buffer M Elution Buffer M Sealing Foils 1 5 ml Receiver Tubes Manual Initial steps Plastic to be supplied 2 0 ml Deep Well Plate KF96 Tip Comb for DW 200 ul Elution Plate 1 x 96 preparations 7450300150 15 ml 150 mg 30 ml 2 x 1 2 ml working solution 3x2ml 2 x 1 1 ml working solutio

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