InviMag SalivaGene DNA Kit/ KFmL User manual
Contents
1. Incubate for 10 min at 37 C while continuously shaking Afterwards add 20 ul Proteinase K and incubate for 20 min at 56 C while continuously shaking Continue with step 2 of the manual During lysis prefill all Tube Stripes with required buffers and appropriate volumes 1b Human DNA Transfer 500 ul stabilized sample into a 1 5 ml reaction tube add 20 ul Proteinase K and incubate for 20 min at 56 C while continuously shaking Continue with step 2 During lysis prefill all Tube Stripes with required buffers and appropriate volumes 2 After the lysis step transfer the whole sample from step 1a or 1b into the Tube Stripe A containing 200 ul Binding Buffer A and 20 ul MAP B and select the program InviMag Salivagene DNA KFmL DNA binds to magnetic particles Magnetic separation Washing of the particle fixed genomic DNA Magnetic separation Elution of genomic DNA Magnetic Separation Pure DNA 14 InviMag SalivaGene DNA Kit KFmL 0615 Protocol I Human DNA extraction from stabilized material Please read the instructions carefully and conduct the prepared procedure Attention Please be aware that you have to prepare the Binding Buffer A see instruction page 10 Lysis external Transfer 500 ul of Saliva DNA Stabilizing Mixture from the SalivaGene Collection Tube into a 1 5 ml reaction tube not provided Add 20 ul of Proteinase K and incubate for 20 min at 56 C while continuously sh2. KingFisher ml tip comb Binding Plate Salivagene A Binding Beginning of step Precollect No Release time hh mm ss 00 00 10 Release speed Fast Mixing pause parameters Pause for manual handling No Mixing time hh mm ss 00 05 00 Mixing speed Slow End of step Postmix No Collect count 4 Collect time s 3 Wash 1 Plate Salivagene B Wash 1 Beginning of step Precollect No Release time hh mm ss 00 00 10 Release speed Fast Mixing pause parameters Pause for manual handling No Mixing time hh mm ss 00 01 30 Mixing speed Medium End of step Postmix No Collect count 3 Collect time s 2 Wash 2 Plate Salivagene C Wash 2 Beginning of step Precollect No Release time hh mm ss 00 00 10 Release speed Fast Mixing pause parameters Pause for manual handling No Mixing time hh mm ss 00 01 00 Mixing speed Medium End of step Postmix No Collect count 3 Collect time s 2 Wash 3 Plate Salivagene D Wash 3 Beginning of step Precollect No Release time hh mm ss 00 00 10 Release speed Medium Mixing pause parameters Pause for manual handling No Mixing time hh mm ss 00 01 00 Mixing speed Medium End of step Postmix No InviMag SalivaGene DNA Kit KFmL 0615 Collect count 3 Collect time s 2 Drying Plate Salivagene D Wash 3 Dry time hh mm ss 00 03 30 Tip position Outside well tube Elutio
3. Pipetting genomic DNA through small tip openings causes shearing or nicking One way to decrease shearing of genomic DNA is to use special tips that have wide openings designed for pipetting genomic DNA 21 InviMag SalivaGene DNA Kit KFmL 0615 Ordering information Product Package size Catalogue No InviMag SalivaGene DNA Kit KFmL 15 preps 2435160100 InviMag SalivaGene DNA Kit KFmL 75 preps 2435160200 InviMag SalivaGene DNA Kit KFmL 300 preps 2435160400 InviMag SalivaGene DNA Kit KF96 KFflex 96 1x 96 preps 7435300100 InviMag SalivaGene DNA Kit KF96 KFflex 96 5x 96 preps 7435300200 PSP SalivaGene DNA Kit 5 preparations 1035200100 PSP SalivaGene DNA Kit 50 preparations 1035200200 PSP SalivaGene DNA Kit 250 preparations 1035200300 SalivaGene Collection Module II 5 container 1035210600 SalivaGene Collection Module II 50 container 1035210700 SalivaGene Buccal Swab 5 pieces 1035230100 SalivaGene Buccal Swab 50 pieces 1035230200 Ordering information KingFisher mL and consumables from Thermo Scientific 5400050 KingFisher mL Magnetic Particle Processor 100 240 V 50 60 Hz 97002111 KingFisher mL tip comb 800 pcs 97002121 KingFisher mL tube 900 pcs 20x45 pcs 97002131 KingFisher mL Combi 60 tubes and tip combs for 60 samples 97002141 KingFisher mL Combi 240 tubes and tip combs for 240 samples Possible suppliers for lsopropanol Fa Carl Roth 5 f 2 Propanol Fa Applichem Fa Sigma Rotipuran
4. Putting some grains of sugar on the tongue helps to produce saliva and does not interfere with the stabilization Unscrew lid from Collection Tube and put it aside for further use Rub cheeks against teeth intensely for 30 sec Collect saliva to indicated fill level avoid making and measuring air bubbles Unscrew and discard lid from Stabilizer Tube Pour Saliva DNA Stabilizer reagent into Collection Tube Screw lid tightly onto the Collection Tube again Shake tube for 15 sec to mix saliva and Saliva DNA Stabilizer reagent 12 InviMag SalivaGene DNA Kit KFmL 0615 PSP Treatment of SalivaGene Buccal Swab Intended Use Collection of human oral samples for DNA stabilization and extraction using the SalivaGene DNA Extraction Kits The DNA in SalivaGene stabilized samples is stable for 1 year at room temperature within the expiry date The product is intended for use under supervision of professionals only such as technicians physicians and biologist trained in molecular biological techniques Contents Swab Stabilizer tube label for donor description The Stabilizer tube contains approx 2 ml of liquid DNA Stabilizer reagent Limitations of the procedure Do not eat drink smoke or chew gum at least 30 min prior to swab collection Ensure the swab tip does not come into contact with any surface prior to collection Put the Stabilizer tube upright to prevent the liquid inside the tube from spilling Be sure to move the
5. 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 SalivaGene DNA Kit KFmL 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 infectious and be handled and discarded according to local safety regulations European Community risk and safety phrases for the components of the InviMag SalivaGene DNA Kit KFmL to which they apply are listed below as follows Wash Buffer warming H302 312 332 412 EU032 P273 Proteinase K SalivaGene DNA Stabilizer danger danger H315 319 334 335 P280 305 351 338 310 405 H319 P305 351 338 H319 Causes serious eye irritation H302 Harmful if swallowed H312 Harmful in contact with skin H319 Causes serious eye irritation H332 Harmful if inhaled H412 Harmful to aquatic life with long lasting effects EUH032 Contact with acids liberates very toxic gas H315 Causes skin irritation H334 May cause allergy or asthma symptoms or breathing difficulties if inhaled H335 May cause respiratory irritation P305 P351 P338 If in eyes Rinse cautiously with water for several minutes Remove contact lenses if present and easy to do Continue
6. 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 SalivaGene DNA Kit KFmL 0615 Contents Kit content of the InviMag SalivaGene DNA Kit KFmL 3 Kit content of the InviMag SalivaGene DNA Kit KFmL w o plastic 4 Content of SalivaGene Stabilization Sets 5 Symbols 5 Storage 5 Quality control 6 Intended use of the InviMag SalivaGene DNA Kit KFmL 6 Product use limitation 6 Safety information 7 Product characteristic of the PSP SalivaGene DNA Kit KFmL 8 Sampling and sample storage 9 Principle and procedure 9 Yield and quality of DNA 9 Important points before starting a protocol 10 Preparing reagents and buffers 10 Reagents and equipment to be supplied by user 11 PSP Treatment with SalivaGene Collection Module II 12 PSP Treatment of SalivaGene Buccal Swab 13 Scheme for KingFisher mL instrument 14 Protocol I Human DNA extraction from stabilized material 15 Protocol Il Bacterial DNA extraction from stabilized material 16 For self programming of the KFmL instrument 17 Troubleshooting 19 Appendix 20 General notes on handling DNA 21 Ordering information 22 2 InviMag SalivaGene DNA Kit KFmL 0615 Kit content of the InviMag SalivaGene DNA Kit KFmL Store dissolved Proteinase K and Lysozyme at 20 C Store all other kit components at room temperature RT Catalogue No Lysozyme Binding B
7. by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times Add 2 ml ddH20 to the tube with Proteinase K mix thoroughly until completely dissolving and store at 20 C Add 1 ml ddH2O to each tube with Lysozyme mix thoroughly and store the tube at 20 C Add 30 ml of 96 100 ethanol to the bottle Wash Buffer I mix thoroughly and store with tightly closed cap Add 42 ml of 96 100 ethanol to each bottle Wash Buffer Il mix thoroughly and store with tightly closed cap 300 DNA extractions Add 56 ml 99 7 Isopropanol to the Binding Buffer A Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times Add 7 ml ddH20 to the tube with Proteinase K mix thoroughly until completely dissolving and store at 20 C Add 7 ml ddH20 to the tube with Lysozyme mix thoroughly and store the tube at 20 C Add 80 ml of 96 100 ethanol to the bottle Wash Buffer I mix thoroughly and store with tightly closed cap Add 140 ml of 96 100 ethanol to each bottle Wash Buffer Il mix thoroughly and store with tightly closed cap 10 InviMag SalivaGene DNA Kit KFmL 0615 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 These are available online in convenient and compact PDF format
8. is responsible to validate the performance of the STRATEC Molecular product for any particular use STRATEC Molecular does not provide for validation of performance characteristics of the product with respect to specific applications STRATEC Molecular products may be used e g in clinical diagnostic laboratory systems conditioned upon the complete diagnostic system of the laboratory the laboratory has been validated pursuant to CLIA 88 regulations in the U S or equivalents in other countries 6 InviMag SalivaGene DNA Kit KFmL 0615 All products sold by STRATEC Molecular are subject to extensive quality control procedures according to ISO 9001 2000 and ISO EN 13485 and are warranted to perform as described herein Any problems incidents or defects shall be reported to STRATEC Molecular immediately upon detection thereof The chemicals and the plastic parts are for laboratory use only they must be stored in the laboratory and must not be used for purposes other than intended The product with its contents is unfit for consumption Safety information When and while working with chemicals always wear a suitable lab coat disposable gloves and protective goggles Avoid skin contact Adhere to the legal requirements for working with biological material For more information please consult the appropriate material safety data sheets MSDS These are available online in convenient and compact PDF format at www stratec com for each STRATEC
9. rinsing P273 Avoid release to the environment P280 Wear protective gloves protective clothing eye protection face protection 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 in USA 1 800 535 5053 7 InviMag SalivaGene DNA Kit KFmL 0615 Product characteristic of the PSP SalivaGene DNA Kit KFmL Starting material 500 ul of stabilization buffer up to 10 pg 23 min SalivaGene DNA Stabilizer depends on kind of saliva mix 1 1 starting material Rout YSIS The InviMag SalivaGene DNA Kit KFmL uses the PSP Technology Preanalytical Sample Processing a patented cost efficient technology and is a modular designed system for collection stabilization storage and transportation of saliva samples with DNA purification The system combines the use of a prefilled SalivaGene Collection Tube for saliva sample collection the storage and stabilization of saliva specimen without any degradation of the DNA during transportation including the prelysis of bacteria and a very efficient and fast isolation about 23 min of high quality total DNA from up to 500 ul mixture using the KFmL instrument The kit is designed for isolation of DNA from host organism as well as for isolation of DNA from pathogen microorganisms After sample collection the saliva sample is t
10. the workstation memory 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 1 for assay development as this software version includes the correct adjustments for this plate It is highly recommended to use Thermo Microtiter Deep Well plates with KF96 KFflex96 workstations to ensure the best purification result PC requirements for KingFisher Software 3 1 PC requirements Interface Serial communication port via a RS 232 full duplex interface Microsoft Windows 2000 Supparied ope tating systems Microsoft Windows XP Professional Disk space 500 MB free disk space Processor Intel Pentium gt 700 MHz recommended Memory 220 MB RAM recommended Serial ports available 1 Pointing device Mouse or equivalent is necessary CD ROM drive 1 SVGA monitor with at least 1024 x 768 resolution and at least MAOTA ceVaireet IDEs a 16 bit color environment Microsoft Windows 2000 Service Pack 4 or greater Service packs installed Microsoft Windows XP Professional Service Pack 2 or greater Browser Microsoft Internet Explorer 6 0 or greater installed If you do not have the correct Service Packs installed you can download them from the Microsoft web pages http www microsoft com 20 InviMag SalivaGene DNA Kit KFmL 0615 General notes on handling DNA Sta
11. TA degraded incorrect storage of starting ensure the sample is harvested and stored correctly or sheared material DNA old material avoid repeated thawing and freezing cycles of the material Old samples often contain degraded DNA problems ethanol in the eluted DNA verify if the recommended dry time was reached with increase drying time for the elimination of ethanol in the run file subsequent applications salt in the eluate Wash Buffer should be stored and used at RT e g in PCR make sure that no precipitates are visible within the Washing reduced sensitivity of amplification reaction Buffers If any precipitations are visible dissolve them by carefully warming up to 30 C adjust the volume of eluate added as template in the amplification reaction A260 A2g0 rati for purified DNA is low insufficient cell lysis and protein degradation due to decreased Proteinase K Lysozyme activity insufficient lysis due to incomplete mixing with lysis buffer see above under low amount of DNA see above under low amount of DNA 19 InviMag SalivaGene DNA Kit KFmL 0615 Appendix KingFisher Software 3 1 The KingFisher Software 3 1 was used to create assay files for the KFmL KF96 and KFflex96 instruments The respective assay file can either be transferred onto the KingFisher workstation or be started directly from within the Bindlt software Keep in mind that directly run assay files are not stored in
12. aking or alternatively flick the tube every 5 min Note If you have to archive a SalivaGene sample e g for forensic purposes store the residual sample not needed for preparation at 20 C The sample is stable for several years under these conditions Prepare the Tube Stripes with the appropriate buffers and volumes needed during the lysis process see below for detailed information During lysis prepare the Tube Stripes with the provided buffers and appropriate volumes See below for detailed information Important Mix the bottle with the MAP B by vigorously vortexing Tube Stripe A Transfer 200 ul Binding Buffer A and 20 ul MAP B into Tube Stripe A Tube Stripe B Transfer 500 ul Wash Buffer Tube Stripe C Transfer 600 ul Wash Buffer Il Tube Stripe D Transfer 600 ul Wash Buffer Il Tube Stripe E Transfer 200 ul Elution Buffer Starting a run After lysis transfer the lysed sample into the Tube Stripe A containing 200 ul Binding Buffer A and 20 ul MAP B Start the purification by selecting the assay file InviMag Salivagene DNA KFmL and press the START button The DNA will bind to the magnetic beads and will then be transported through the different washing solutions before the elution will take place in Tube Stripe E The eluate can finally be transferred into the provided Receiver Tubes 15 InviMag SalivaGene DNA Kit KFmL 0615 Protocol II Bacterial DNA extraction from stabilized material Please read the
13. andling of potentially infectious samples The procedure is designed to prevent sample to sample cross contaminations Due to the high purity the isolated genomic DNA is ready to use for a broad panel of downstream applications see below or can be stored at 20 C for subsequent use PCR Real Time PCR Analysis RFLP AFLP Analysis Restriction Enzyme Digestion HLA Typing Pathogen Detection O O OO The PCR method is covered by U S Patents 4 683 195 and 4 683 202 owned by Hoffmann LaRoche Inc The purchase of the InviMag SalivaGene DNA Kit KFmL cannot be construed as an authorization or implicit license to practice PCR under any patents held by Hoffmann LaRoche Inc 8 InviMag SalivaGene DNA Kit KFmL 0615 Sampling and sample storage Starting material The amount of starting material used in InviMag SalivaGene DNA Kit KFmL procedure can vary slightly depending on the amount of DNA present in the sample Specific guidance for starting amounts is given in the individual protocols The protocol works with aliquots of the transport media in which fresh saliva samples are collected The DNA in SalivaGene stabilized samples is stable for 1 year at room temperature within the expiry date The aliquots of the stabilization buffer may be processed on the same day as collection or stored for future processing For long term storage store the sample at 20 C STRATEC Molecular will be released of its responsibilities if o
14. at www sitratec com under each STRATEC Molecular kit and kit component Thermomixer 56 C human bacteria 37 C bacteria only Measuring cylinder 250 ml Disposable gloves Pipette and pipette tips Vortexer ddH O 96 100 ethanol 1 5 ml reaction tube s Isopropanol 0O O 0 0 0 0 0 0 O The InviMag SalivaGene DNA Kit KFmL is validated with 2 Propanol Rotipuran gt 99 7 p a ACS ISO Order no 6752 from Carl Roth Possible suppliers for lsopropanol Fa Carl Roth 2 Propanol Fa Applichem Fa 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 11 InviMag SalivaGene DNA Kit KFmL 0615 PSP Treatment with SalivaGene Collection Module II Intended Use Collection of human saliva samples for DNA stabilization and extraction using the SalivaGene DNA Extraction Kits The DNA in SalivaGene stabilized samples is stable for 1 year at room temperature within the expiry date The product is intended for use under supervision of professionals only such as technicians physicians and biologist trained in molecular biological techniques Contents Collection tube Stabilizer tube The stabilizer tube contains approx 2 ml of liquid Saliva DNA Stabilizer reagent Limitations of the procedure Do not eat drink smoke or chew gum at least 30 min prior to saliva collection Collecting 2 ml of saliva may require several minutes
15. bottle Wash Buffer I mix thoroughly and store with tightly closed cap Add 42 ml of 96 100 ethanol to each bottle Wash Buffer II mix thoroughly and store with tightly closed cap 300 extractions 2435160400 for 7 ml working solution 24 ml final volume 80 ml 7 ml for 7 ml working solution 80 ml final volume 160 ml 2 x 60 ml final volume 2 x 200 ml 80 ml 4x15 20x 15 20 x 15 1 Add 56 ml 99 7 Isopropanol to the Binding Buffer A Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times Add 7 ml ddH20O to the tube with Proteinase K mix thoroughly until completely dissolving and store at 20 C Add 7 ml ddH20 to the tube with Lysozyme mix thoroughly and store the tube at 20 C Add 80 ml of 96 100 ethanol to the bottle Wash Buffer I mix thoroughly and store with tightly closed cap Add 140 ml of 96 100 ethanol to each bottle Wash Buffer Il mix thoroughly and store with tightly closed cap InviMag SalivaGene DNA Kit KFmL 0615 Kit content of the InviMag SalivaGene DNA Kit KFmL w o plastic Store dissolved Proteinase K and Lysozyme at 20 C Store all other kit components at room temperature RT Catalogue No Lysozyme Binding Buffer A MAP Solution B Proteinase K Wash Buffer Wash Buffer Il Elution Buffer Lysozyme MAP B 1 5 ml Receiver Tubes Manual Initial steps Plastic to be supplied by user s
16. d in different downstream applications Yield and quality of DNA The amount of purified DNA in the InviMag SalivaGene DNA Kit KFmL procedure from different samples depends on the sample type sample source transport storage and age Yield and quality of isolated DNA are suitable for any molecular diagnostic detection system The diagnostic tests should be performed accordingly to the manufacturer s specifications 9 InviMag SalivaGene DNA Kit KFmL 0615 Important points before starting a protocol General handling 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 you have to notify STRATEC Molecular in writing with immediate effect upon inspection thereof If buffer bottles are damaged contact the STRATEC Molecular Technical Services or your local distributor In case of liquid spillage refer to Safety Information see page 7 Do not use damaged kit components since their use may lead to poor kit performance o Always change pipette tips between liquid transfers To avoid cross contaminations we recommend the use of aerosol barrier pipette tips o 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 A
17. ee order information KingFisher mL Tip Combs KingFisher mL Tube Strips 15 extractions 2435160150 for 500 ul working solution 4x1ml ready to use 0 5 ml for 500 ul working solution 7 5 ml final volume 15 ml 18 ml final volume 60 ml 2ml for 500 ul working solution 500 ul 15 1 Add 500 ul ddH20 to the tube with Proteinase K mix thoroughly until completely dissolving and store at 20 C Add 500 ul ddH20 to the tube with Lysozyme mix thoroughly and store the tube at 20 C Add 7 5 ml of 96 100 ethanol to the bottle Wash Buffer I mix thoroughly and store with tightly closed cap Add 42 ml of 96 100 ethanol to each bottle Wash Buffer II mix thoroughly and store with tightly closed cap 75 extractions 2435160250 for 2 x 1 ml working solution 9 ml final volume 30 ml 2x1 ml for 2 ml working solution 30 ml final volume 60 ml 2x 18 ml final volume 2 x 60 ml 30 ml for 2 x 1 ml working solution 2x1 ml 5x15 1 Add 21 ml 99 7 Isopropanol to the Binding Buffer A Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times Add 2 ml ddH20 to the tube with Proteinase K mix thoroughly until completely dissolving and store at 20 C Add 1 ml ddH20 to each tube with Lysozyme mix thoroughly and store the tube at 20 C Add 30 ml of 96 100 ethanol to the bottle Wash Buffer I mix thoroughly and store with tight
18. gt 99 7 p a ACS ISO 2 Propanol f r die Molekularbiologie 2 Propanol Order no 6752 Order no A3928 Order no 59304 1 L F 22 InviMag SalivaGene DNA Kit KFmL 0615 stratecee molecular STRATEC Molecular GMbH Robert R ssle Str 10 13125 Berlin Germany Phone 49 30 94 89 29 01 Fax 49 30 94 89 29 09 E mail info berlin stratec com www stratec com 1G3g02 06 2015
19. instructions carefully and conduct the prepared procedure Attention Please be aware that you have to prepare the Binding Buffer A see instruction page 10 Lysis external Transfer 500 ul Saliva DNA Stabilizing Mixture from the SalivaGene Collection Tube into a 1 5 ml reaction tube not provided Add 20 ul of Lysozyme vortex for 5 s and incubate the mixture for 10 min at 37 C while continuously shaking or flicking the tube from time to time Afterwards add 20 ul Proteinase K and incubate for an additional 20 min at 56 C while continuously shaking or every 5 minutes flicking the tube Note If you have to archive a SalivaGene sample e g for forensic purposes just freeze the rest you don t use for preparation The rest of the stabilized sample can be stored at 20 TC for years During lysis prepare the Tube Stripes with the provided buffers and appropriate volumes See below for detailed information Important Mix the bottle with the MAP B by vigorously vortexing Tube Stripe A Transfer 200 ul Binding Buffer A and 20 ul MAP B into Tube Stripe A Tube Stripe B Transfer 500 ul Wash Buffer Tube Stripe C Transfer 600 ul Wash Buffer Il Tube Stripe D Transfer 600 ul Wash Buffer Il Tube Stripe E Transfer 200 ul Elution Buffer Starting a run After lysis transfer the lysed sample into the Tube Stripe A containing 200 ul Binding Buffer A and 20 ul MAP B Start the protocol by selecting the assay file InviMag Sa
20. lecular warrants the correct function of the InviMag SalivaGene DNA Kit KFmL for applications as described in this manual Purchaser must determine the suitability of the product for its particular use Should any product fail to perform the applications as described in the manual STRATEC Molecular will check the lot and if STRATEC Molecular investigates a problem in the lot STRATEC Molecular will replace the product free of charge 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 SalivaGene DNA Kit KFmL 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 SalivaGene DNA Kit KFmL 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 2907 or contact your local distributor Intended use The InviMag SalivaGene DNA Kit KFmL is suitable for reproducible purification of total DNA from
21. livaGene Swab Collection Tube prefilled with DNA Stabilizer 500 ul SalivaGene DNA Stabilizer in seas SalivaGene Buccal Swab 1035230100 1035230200 Buccal Swabs 50 pieces SalivaGene Swab Collection Tube prefilled with DNA Stabilizer 500 ul SalivaGene DNA Stabilizer SU cea Symbols pall Manufacturer Lot number T o Catalogue number Expiry date Consult operating instructions Temperature limitation SHEE Do not reuse Storage All buffers and kit components except the dissolved Proteinase K and the Lysozyme of the InviMag SalivaGene DNA Kit KFmL should be stored sealed and dry at room temperature and are stable for at least 12 months under these conditions Dissolved Proteinase K and Lysozyme must be stored at 20 C Repeated freezing and thawing cycles will reduce the activity of Proteinase K and Lysozyme dramatically Dividing of dissolved Proteinase K and Lysozyme into aliquots and storage at 20 C is recommended Wash Buffer I and II charged with ethanol should be appropriate sealed and stored at room temperature The MAP B magnetic beads should be stored at 2 8 C Before use make sure that all components have room temperature If any precipitates are visible within the provided solutions solve them by carefully warming up to 30 C Room temperature RT is defined as range from 15 30 C 5 InviMag SalivaGene DNA Kit KFmL 0615 Quality control and product warranty STRATEC Mo
22. livagene DNA KFmL and press the START button The DNA will bind to the magnetic beads and will be transported through the different washing buffers before the elution is performed in Tube Stripe E The eluate can finally be transferred into the provided Receiver Tubes after the assay has been finished 16 InviMag SalivaGene DNA Kit KFmL 0615 For self programming of the KFmL instrument Protocol Properties Software Version Bindlt 3 1 Name InviMag Salivagene DNA KFmL Protocol template version 3 1 Instrument type KingFisher mL Creator STRATEC Molecular GmbH Description KFmL protocol Thermo Electron for isolation of genomic mitochondrial and or bacterial DNA from saliva samples human stored in Saligene Stabilization Tubes with InviMag SalivaGene DNA Kit KFmL Kit InviMag SalivaGene DNA Kit KFmL Layout Data Tray plate type KingFisher tubestrip 1000 ul Tube A Binding Reagents Name Lysed Sample Volume ul 520 Type Sample Name MAP B Volume pl 20 Type Reagent Name Binding Buffer A Volume ul 200 Type Reagent Tube B Wash 1 Reagents Name Wash Buffer Volume yl 500 Type Reagent Tube C Wash 2 Reagents Name Wash Buffer II Volume yl 600 Type Reagent Tube D Wash 3 Reagents Name Wash Buffer II Volume yl 600 Type Reagent Tube E Elution Reagents Name Elution Buffer Volume pl 200 Type Reagent Steps Tip1
23. ly closed cap Add 42 ml of 96 100 ethanol to each bottle Wash Buffer Il mix thoroughly and store with tightly closed cap 15 5x15 300 extractions 2435160450 for 7 ml working solution 24 ml final volume 80 ml 7 ml for 7 ml working solution 80 ml final volume 160 ml 2x 60 ml final volume 2 x 200 ml 80 ml for2x1ml working solution 2x1 ml 20x15 1 Add 56 ml 99 7 Isopropanol to the Binding Buffer A Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times Add 56 ml 98 100 lsopropanol to the Binding Buffer A Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times Add 7 ml ddH20 to the tube with Proteinase K mix thoroughly until completely dissolving and store at 20 C Add 7 ml ddH O to the tube with Lysozyme mix thoroughly and store the tube at 20 C Add 80 ml of 96 100 ethanol to the bottle Wash Buffer I mix thoroughly and store with tightly closed cap Add 140 ml of 96 100 ethanol to each bottle Wash Buffer Il mix thoroughly and store with tightly closed cap 4x15 20x15 InviMag SalivaGene DNA Kit KFmL 0615 Content of SalivaGene Stabilization Sets SalivaGene Collection Module II 1035210600 1035210700 DNA Stabilizer 2 ml SalivaGene DNA Stabilizer prefilled in 5 pieces 50 pieces a Transport Tube SalivaGene Collection Tube provided as Set 50 pieces Sa
24. n Plate Salivagene E Elution Beginning of step Precollect No Release time hh mm ss 00 00 10 Release speed Medium Mixing pause parameters Pause for manual handling No Mixing time hh mm ss 00 10 00 Mixing speed Slow End of step Postmix No Collect count 4 Collect time s 3 Bead Removal Plate Salivagene D Wash3 Release time hh mm ss 00 00 30 Release speed Fast InviMag SalivaGene DNA Kit KFmL 0615 Troubleshooting Problem Cause Comments and suggestions amount insufficient cell lysis due to repeat the DNA purification procedure with a new sample and of DNA decreased Proteinase K freshly prepared Proteinase K Lysozyme stock solution Lysozyme activity be sure to make aliquots and store them at 20 C insufficient lysis due to repeat the DNA purification procedure with a new sample Be insufficient mixing with sure to mix the sample and SalivaGene DNA Stabilizer lysis buffer SalivaGene DNA immediately Stabilizer low percentage alcohol used repeat purification procedure with a new sample incomplete elution increase incubation time with Elution Buffer to 5 10 min or increase the elution volume to 200 ul This will however reduce the final DNA concentration Make sure to adjust modification in the KingFisher software pH of water incorrect acidic low pH may reduce DNA yield Ensure that the pH of the water is at least 7 0 or use Elution Buffer contains 10 mM Tris HCl but no ED
25. ransferred in the SalivaGene Collection Tube prefilled with SalivaGene DNA Stabilizer The raw material is lysed under these specific conditions The SalivaGene DNA Stabilizer causes an inactivation of DNases and thereby prevents the degradation of DNA preserves the microorganism titer and prelyses bacteria The transport of the stabilized DNA can be carried out in the SalivaGene DNA Stabilizer containing tubes without cooling during 6 weeks at ambient temperature The sample can also be stored in this buffer at 20 C for a long term storage For the DNA extraction process an aliquot or the whole volume is used The residual sample can be stored and used for further extractions A 500 ul aliquot of the stabilized sample is used in the PSP SalivaGene DNA isolation procedure At first the proteins are degraded with Proteinase K followed by a binding of the DNA to magnetic beads while contaminants like potent inhibitors and proteins are rejected and washed out leaving the pure DNA to be eluted in either water or the buffer provided within the kit For reproducible and high yields appropriate sample storage is essential Yields may be varying from sample to sample depending on factors such as the health of the donor patient medication or sample storage conditions The procedure neither requires a phenol chloroform extraction nor an alcohol precipitation Furthermore the kit requires only minimal interaction by the user allowing safe h
26. rting material This kit is designed for extraction of DNA from saliva and swabs These materials show big variation in DNA contents The purification of some apoptotic DNA is normal 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 compatibility with various downstream applications Damaged DNA could perform poorly in applications such as genomic Southern blotting and long template PCR Storage of DNA A working stock of DNA can be stored at 2 4 C for several weeks For long term storage DNA should be stored at 20 C but storing at 20 C can cause shearing particularly if the DNA is exposed to repeated freeze thaw cycles Note that the solution in which the nucleic acid is eluted in will affect its stability during storage Pure water lacks buffering capacity and an acidic pH may lead to acid hydrolysis Tris or Tris EDTA buffer contains sufficient buffering capacity to prevent acid hydrolysis Drying dissolving and pipetting DNA Avoid over drying genomic DNA after ethanol precipitation It is better to let it air dry than to use a vacuum although vacuum drying can be used with caution Avoid vigorous pipetting
27. stratecee molecular User manual InviM g SalivaGene DNA Kit KFmL for use on KingFisher mL Thermo Fisher Scientific for automated purification of total DNA from SalivaGene stabilized clinical swab mouth brushes amp saliva sample with magnetic beads 2435160X00 pss STRATEC Molecular GmbH D 13125 Berlin re Instruction for InviMag SalivaGene DNA Kit KFmL The InviMag SalivaGene DNA Kit KFmL is used to stabilize and purify human and or bacterial genomic DNA quantitatively by use of magnetic beads on the KingFisher mL instrument The InviMag SalivaGene DNA Kit KFmL is a modular system using a stabilization solution for collection stabilization storage and transportation of saliva material without degradation of DNA for at least 12 month with a very efficient isolation method for high quality DNA The kit is neither suitable for the isolation of DNA from stool samples fungi nor for purification of total RNA Trademarks InviMag SalivaGene Invisorb PSP 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 SalivaGene Invisorb and PSP are registered trademarks of STRATEC Biomedical AG The
28. swab over the entire cheek and to moisten it with saliva Open swab package and remove swab without touching the tip Insert swab into the mouth and rub swab tip firmly against the inner cheeks for about 30 seconds on each side Unscrew lid from Stabilizer tube and put it aside for further use Insert swab into the Stabilizer tube Break swab tip at breaking point Discard the broken handle part Screw lid tightly onto the Stabilizer tube again Gently shake tube for 15 sec to mix buccal cells and DNA Stabilizer reagent Label Stabilizer tube with donor name and collection date using provided label 13 InviMag SalivaGene DNA Kit KFmL 0615 Scheme of the InviMag SalivaGene DNA Kit KFmL Binding Tube A Elution Tube E Please carefully read protocols prior starting with the preparation Transfer 500 ul stabilized sample into a 1 5 ml reaction tube and add the appropriate lysis reagent PK and or Lysozyme see 1a or 1b During lysis prefill the Tube Stripes with the needed buffers as described below Washing Tube B Washing Tube C Washing Tube D Add 200 ul Binding Buffer A follow preparing instructions and 20 ul MAP B Add 500 ul Wash Buffer Add 600 ul Wash Buffer II Add 600 ul Wash Buffer II Add 200 ul Elution Buffer _ gt ZE a Gat 1a Bacterial DNA Transfer 500 ul stabilized sample into a 1 5 ml reaction tube and add 20 ul Lysozyme
29. ther sample materials than described in the Intended Use are processed or if the sample preparation protocols are changed or modified Principle and procedure The InviMag SalivaGene DNA Kit KFmL procedure comprises the following steps o Collection of the material using a SalivaGene Collection Set o Lysis under non chaotropic conditions at elevated temperature in Stabilization Buffer SalivaGene DNA Stabilizer Protein digestion Proteinase K Binding of the genomic DNA to magnetic beads MAP B Washing of the beads and elimination of contaminants and ethanol Elution of pure genomic DNA O O 0 This manual contains 2 protocols according to the different requirements of the starting materials used Lysis Samples are lysed under non chaotropic conditions at elevated temperature in the Stabilization Buffer SalivaGene DNA Stabilizer Protein digestion Proteins will be digested in the lysed samples in the presence of Proteinase K Binding genomic DNA By adding Binding Buffer A to the lysate optimal binding conditions are adjusted Each lysate is then applied to magnetic beads and the genomic DNA is bound to the beads Removing residual contaminations Contaminants are efficiently washed away using Wash Buffer I and II while the genomic mitochondrial and bacterial DNA remains bound to the magnetic beads Elution of pure genomic DNA The isolated DNA is eluted from the magnetic beads using Elution Buffer and can be use
30. uffer A MAP Solution B Proteinase K Wash Buffer Wash Buffer Il Elution Buffer KingFisher mL Tip Combs KingFisher mL Tube Strips 1 5 ml Receiver Tubes Manual Initial steps 15 extractions 2435160100 for 500 ul working solution 4x1ml ready to use 0 5 ml for 500 ul working solution 7 5 ml final volume 15 ml 18 ml final volume 60 ml 2 ml 3 15 15 1 Add 500 ul ddH20 to the tube with Proteinase K mix thoroughly until completely dissolving and store at 20 C Add 500 ul ddH20 to the tube with Lysozyme mix thoroughly and store the tube at 20 C Add 7 5 ml of 96 100 ethanol to the bottle Wash Buffer I mix thoroughly and store with tightly closed cap Add 42 ml of 96 100 ethanol to each bottle Wash Buffer II mix thoroughly and store with tightly closed cap 75 extractions 2435160200 for 2x1 ml working solution 9 ml final volume 30 ml 2x1 ml for 2 ml working solution 30 ml final volume 60 ml 2x18 ml final volume 2 x 60 ml 30 ml 15 5x15 5x15 1 Add 21 ml 99 7 lsopropanol to the Binding Buffer A Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times Add 2 ml ddH20 to the tube with Proteinase K mix thoroughly until completely dissolving and store at 20 C Add 1 ml ddH20 to each tube with Lysozyme mix thoroughly and store the tube at 20 C Add 30 ml of 96 100 ethanol to the
31. up to 500 ul stabilization media of saliva samples in forensic human identity genetic biosecurity and pathogen analyses The purification is very efficient and the isolated DNA performs well in downstream applications such as quantitative PCR and STR analysis with high signal to noise ratios The system consists of two modules one collection set and one extraction module useable independently or in combination THE PRODUCT IS INDENTED FOR USE BY PROFESSIONALS ONLY SUCH AS TECHNICIANS PHYSICIANS AND BIOLOGISTS TRAINED IN MOLECULAR BIOLOGICAL TECHNIQUES It is designed to be used with any downstream application employing enzymatic amplification or other enzymatic modifications of DNA followed by signal detection or amplification Any diagnostic results generated by using the sample preparation procedure in conjunction with any downstream diagnostic assay should be interpreted with regard to other Clinical or laboratory findings To minimize irregularities in diagnostic results adequate controls for downstream applications should be used Product use limitation The kit is neither validated for the isolation of genomic DNA from serum plasma fungi parasites nor for the isolation and purification of total RNA The included chemicals are only useable once Differing of starting material or flow trace may lead to inoperability therefore neither a warranty nor guarantee in this case will be given neither implied nor express The user
32. void microbial contamination of the kit reagents To minimize the risk of infections from potentially infectious material we recommend working under laminar airflow until the samples are lysed o This kit should only be used by trained personnel O O 0 0 Preparing reagents and buffers Before starting a run equilibrate all reagents to room temperature Where necessary gently mix and redissolve any precipitates by warming up to 30 C Swirl gently to avoid foaming 1 Add the needed amount of ddH O to reaction tube with Proteinase K see below Vortex for 5 s Repeat this step for the Lysozyme using Lysozyme Buffer 2 Add the needed amount of ethanol to Wash Buffer and Wash Buffer II If a KFmL instrument is used for isolation the protein digestion has to be done on an additional themocycler Adjust a thermomixer to 56 C human sample If bacterial DNA should be isolated a previous incubation step at 37 C is needed 15 DNA extractions Add 500 ul ddH20 to the tube with Proteinase K mix thoroughly until completely dissolving and store at 20 C Add 500 ul ddH20 to the tube with Lysozyme mix thoroughly and store the tube at 20 C Add 7 5 ml of 96 100 ethanol to the bottle Wash Buffer I mix thoroughly and store with tightly closed cap Add 42 ml of 96 100 ethanol to each bottle Wash Buffer Il mix thoroughly and store with tightly closed cap 75 DNA extractions Add 21 ml 99 7 Isopropanol to the Binding Buffer A Mix
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