InviMag SalivaGene DNA Kit/ KF96 User manual
Contents
1. 60 ml 20 2x5 10 1 Add 84 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 10 5 ml ddHzO to the bottle with Proteinase K mix thoroughly until completely dissolving and store the tubes at 20 C We recommend to make aliquots and store them at 20 C Add 10 5 ml ddH20 to the bottle with Lysozyme mix thoroughly and store the tubes at 20 C We recommend to make aliquots and store them at 20 C Add 80 ml of 96 100 ethanol to each bottle Wash Buffer I mix thoroughly and store with tightly closed cap at RT Add 140 ml of 96 100 ethanol to each bottle Wash Buffer Il mix thoroughly and store with tightly closed cap at RT InviMag SalivaGene DNA Kit KF96 0615 Kit content of the InviMag SalivaGene DNA Kit KF96 w o plastic Store lyophilized Lysozyme at 2 8 C Store dissolved Proteinase K and Lysozyme at 20 C Store all other kit components at room temperature RT Catalogue No Lysozyme Binding Buffer A Proteinase K MAP Solution B Wash Buffer Wash Buffer Il Elution Buffer Sealing Foils Manual Initial steps Plastic to be supplied by user see order information KF 96 Tip Comb for DW magnets 200 ul Elution Plate 2 0 ml Deep Well Plate 1x 96 extractions 435060150 for 2 x 1 1 ml working solution 9 ml final volume 30 ml for 2 x 1 1 ml working solution2. 2x 1 1 ml 30 ml working solution 60 ml 45 ml working solution 150 ml 15 ml 2 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 1 1 ml ddH2O to each tube with Proteinase K mix thoroughly until completely dissolving and store the tubes at 20 C Add 1 1 ml ddH2O to each tube with Lysozyme mix thoroughly and store the tubes at 20 C Add 30 ml of 96 100 ethanol to the bottle Wash Buffer I mix thoroughly and store with tightly closed cap at RT Add 105 ml of 96 100 ethanol to each bottle Wash Buffer II mix thoroughly and store with tightly closed cap at RT 5x 96 extractions 7435060250 for 10 5 ml working solution 36 ml final volume 120 ml for 10 5 ml working solution 10 5 ml 2 x 80 ml 2 x 160 ml working solution 3 x 60 ml 3 x 200 ml working solution 60 ml 10 1 Add 84 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 10 5 ml ddHzO to the bottle with Proteinase K mix thoroughly until completely dissolving and store the tubes at 20 C We recommend to make aliquots and store them at 20 C Add 10 5 ml ddH2O to the bottle with Lysozyme mix thoroughly and store the tubes at 20 C We recommend to make aliquots and store them at 20 C Add 80 ml
3. C Preheat Postmix Collect count Collect time s Washing Plate 3 Release time speed Tip Plate 19 No 00 00 10 Fast 00 01 30 Fast I uo O o No 00 00 10 Fast 00 01 00 Fast EB o o No 00 00 10 Fast 00 01 00 Fast EB O o 00 03 30 Outside well tube No 00 00 10 Medium 00 10 00 Slow 60 00 00 30 Fast InviMag SalivaGene DNA Kit KF96 0615 For self programming of the KF96 and KFflex96 instrument Human protocol 20 InviMag SalivaGene DNA Kit KF96 0615 Reagent info Lysis Plate Microtiter DW 96 plate Name Well volume pl Total reagent volume pl Type Salivagene Sample 500 Reagent Proteinase K 20 Reagent Washing Plate 1 Microtiter DW 96 plate Name Well volume ul Total reagent volume pl Type Wash Buffer I 500 Reagent Washing Plate 2 Microtiter DW 96 plate Name Well volume pl Total reagent volume pl Type Wash Buffer II 600 Reagent Washing Plate 3 Microtiter DW 96 plate Name Well volume ul Total reagent volume pl Type Wash Buffer II 600 Reagent Hution Plate KingFisher 96 KF plate Name Well volume pl Total reagent volume pl Type Elution Buffer 150 Reagent Tip Plate Microtiter DW 96 plate Name Well volume ul Total reagent volume ul Type Dispensed reagents Lysis Plate Microtiter DW 96 plate Name Step Well volume pl Total reagent volume pl f F Binding Buffer MAP B Adjust Bindik 220 i Solution Steps data Tip Pick Up Lysis Beginni
4. 17 InviMag SalivaGene DNA Kit KF96 0615 Reagent info Lysis Plate Microtiter DW 96 plate Name Well volume pl Total reagent volume pl Type Salivagene Sample 500 Sample Washing Plate 1 Microtiter DW 96 plate Name Well volume pl Total reagent volume pl Type Wash Buffer I 500 Reagent Washing Plate 2 Microtiter DW 96 plate Name Well volume pl Total reagent volume ul Type Wash Buffer II 600 Reagent Washing Plate 3 Microtiter DW 96 plate Name Well volume pl Total reagent volume pl Type Wash Buffer II 600 Reagent Hution Plate KingFisher 96 KF plate Name Well volume pl Total reagent volume pl Type Elution Buffer 150 Reagent Tip Plate KingFisher 96 KF plate Name Well volume pl Total reagent volume pl Type Dispensed reagents Lysis Plate Microtiter DW 96 plate Name Step Well volume pl Total reagent volume pl Proteinase K Add ProteinaseK 20 7 F Binding Buffer MAP B Adjust Budik 220 i Solution Steps data Tip e Pick Up Lysis 1 Beginning of step Mixing heating End of step Add ProteinaseK Reagent s Lysis2 Beginning of step Mixing heating End of step Adjust Binding Reagent s Binding Beginning of step Mixing heating End of step 96 DW tip comb Tip Plate Lysis Plate Precollect Release beads Mixing time speed Heating temperature C Preheat Postmix Collect beads Lysis Plate Message Dispensing volume ul Name Volume u
5. 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 SalivaGene DNA Kit KF96 0615 Table of Content Kit content of the InviMag SalivaGene DNA Kit KF96 3 Kit content of the InviMag SalivaGene DNA Kit KF96 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 KF96 6 Product use limitation 6 Safety information 7 Product characteristic of the InviMag SalivaGene DNA Kit KF96 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 of the InviMag SalivaGene DNA Kit KF96 14 Protocol Human DNA extraction from stabilized material 15 Protocol II Bacterial DNA extraction from stabilized material 16 For self programming of the KF96 and KFflex96 instrument Bacteria protocol 17 For self programming of the KF96 and KFflex96 instrument Human protocol 20 Troubleshooting 23 Appendix 24 General notes on handling DNA 25 Ordering information 26 2 InviMag SalivaGene DNA Kit KF96 0615 Kit content of the InviMag SalivaGene DNA Kit KF96 Store lyophilized
6. eye irritation H302 Harmful if swallowed H312 Harmful in contact with skin H332 Harmful if inhaled H412 Harmful to aquatic life with long lasting effects EUHO32 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 P3054 P351 P338 If in eyes Rinse cautiously with water for several minutes If present remove contact lenses and continue 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 inside of USA 1 800 535 5053 7 InviMag SalivaGene DNA Kit KF96 0615 Product characteristic of the InviMag SalivaGene DNA Kit KF96 500 ul of stabilization buffer up to 10 ug 40 50 min SalivaGene DNA Stabilizer depends on starting material depends on the saliva mix 1 1 used protocol Or 500 ul of swab stabilization SalivaGene DNA Stabilizer The InviMag SalivaGene DNA Kit KF96 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 sample
7. of 96 100 ethanol to each bottle Wash Buffer I mix thoroughly and store with tightly closed cap at RT Add 140 ml of 96 100 ethanol to each bottle Wash Buffer Il mix thoroughly and store with tightly closed cap at RT 2x5 20 InviMag SalivaGene DNA Kit KF96 0615 Content of SalivaGene Stabilization Sets SalivaGene Collection Module II 1035210800 DNA Stabilizer 2 ml SalivaGene DNA Stabilizer prefilled in a ix S0 contaljer Transport Tube SalivaGene Collection Tube provided as Set 10 x 50 container SalivaGene Swab Collection Tube prefilled with DNA Stabilizer 500 ul SalivaGene DNA Stabilizer 10 x 50 pieces SalivaGene Buccal Swab 1035230300 Buccal Swabs 10 x 50 pieces SalivaGene Swab Collection Tube prefilled with DNA Stabilizer 500 ul SalivaGene DNA Stabilizer 10 x 50 pieces Symbols Lot number Catalog number Expiry date Consult operating instructions I Temperature limitation Do not reuse post Manufacturer Storage All buffers and kit components except the dissolved Proteinase K and the Lysozyme of the InviMag SalivaGene DNA Kit KF96 should be stored well sealed and dry at room temperature and are stable for at least 12 months under these conditions Room temperature RT is defined as range from 15 30 C Dissolved Proteinase K and Lysozyme must be stored at 20 C Repeated freezing and thawing will reduce the activity of Proteinase K and Lysozyme dramatically Dividing of Prot
8. plate 1 Add 500 ul Wash Buffer I to a 2 ml Deep Well Plate Washing plate 2 Add 600 ul Wash Buffer Il to a 2 ml Deep Well Plate Washing plate 3 Add 600 ul Wash Buffer Il to a 2 ml Deep Well Plate Elution Plate Add 150 ul Elution Buffer to a KF Elution Plate 7 Choose the program InviMag SalivaGene KFflex96 Bacteria or InviMag SalivaGene KF96 Bacteria on the display of the KingFisher instrument and press the START button 8 Load the prefilled plates into the KingFisher instrument by following the specification given on the display and confirm every following step by pressing the START button 9 When all prefilled plates are loaded into the KingFisher machine press the START button again to execute the assay The run will last about 50 minutes After the first lysis step a pause occurs and 20 ul Proteinase K has to be added to each sample containing cavities of the Binding Plate After Proteinase K distribution press the START button to continue with the lysis After the second lysis step a second pause occurs and 200 ul Binding Buffer A and 20 ul MAP Solution B have to be added to sample containing cavities of the Binding Plate After adding both reagents press the START button again to continue with the assay From this point the instrument will continue without any further user interaction 16 InviMag SalivaGene DNA Kit KF96 0615 For self programming of the KF96 and KFflex96 instrument Bacteria protocol
9. to mix the sample and SalivaGene DNA Stabilizer immediately elongation of the stabilization lysis procedure to overnight significantly increases the overall yield repeat purification procedure with a new sample and or prepare a new bottle of Wash Buffer if a wrong concentration of ethanol was used 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 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 EDTA ensure the sample is harvested and stored at RT avoid repeated thawing and freezing cycles of the material Old material often contains degraded DNA verify if the recommended dry time was reached increase dry time for the elimination of ethanol if necessary Wash Buffer should be stored at and used at RT verify Wash Buffer on the precipitation of salt if there are precipitations dissolve this by carefully warming u to 30 C adjust the volume of eluate added as template in the amplification reaction see above under low amount of DNA see above under low amount of DNA 23 InviMag SalivaGene DNA Kit KF96 0615 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 ca
10. Lysozyme at 2 8 C Store dissolved Proteinase K and Lysozyme at 20 C Store all other kit components at room temperature RT Catalogue No Lysozyme Binding Buffer A Proteinase K MAP Solution B Wash Buffer Wash Buffer Il Elution Buffer 2 0 ml Deep Well Plate KF 96 Tip Comb for DW magnets 200 ul Elution Plate Sealing Foils Manual Initial steps 1x 96 extractions 435060100 for 2 x 1 1 ml working solution 9 ml final volume 30 ml for 2 x 1 1 ml working solution 2x1 1 ml 30 ml working solution 60 ml 45 ml working solution 150 ml 15 ml 4 2 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 1 1 ml ddH2O to each tube with Proteinase K mix thoroughly until completely dissolving and store the tubes at 20 C Add 1 1 ml ddH2O to each tube with Lysozyme mix thoroughly and store the tubes at 20 C Add 30 ml of 96 100 ethanol to the bottle Wash Buffer I mix thoroughly and store with tightly closed cap at RT Add 105 ml of 96 100 ethanol to each bottle Wash Buffer Il mix thoroughly and store with tightly closed cap at RT 5x 96 extractions 7435060200 for 10 5 ml working solution 36 ml final volume 120 ml for 10 5 ml working solution 10 5 ml 2 x 80 ml 2 x 160 ml working solution 3 x 60 ml 3 x 200 ml working solution
11. a vacuum although vacuum drying can be used with caution 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 DNA yield The amount of purified DNA depends on sample source transport conditions storage and age of the sample 25 InviMag SalivaGene DNA Kit KF96 0615 Ordering information Product Package size Catalogue No InviMag SalivaGene DNA Kit KF96 1 x 96 preps 7435060100 InviMag SalivaGene DNA Kit KF96 5 x 96 preps 7435060200 InviMag SalivaGene DNA Kit KF96 w o plastic 1 x 96 preps 7435060150 InviMag SalivaGene DNA Kit KF96 w o plastic 5 x 96 preps 7435060250 SalivaGene Collection Module II 5 container 1035212100 SalivaGene Collection Module II 50 container 1035212200 SalivaGene Collection Module II 125 container 1035212300 SalivaGene Buccal Swab 5 pieces 1035230100 SalivaGene Buccal Swab 50 pieces 1035230200 SalivaGene Buccal Swab 8 x 50 pieces Related products InviMag SalivaGene DNA Kit KFmL 15 preps 2435160100 InviMag SalivaGene DNA Kit KFmL 75 preps 2435160200 InviMag SalivaGene DNA Kit KFmL 300 preps 2435160400 PSP SalivaGene DNA Kit 50 preparations 1035200200 PSP SalivaGene DNA Kit 250 preparations 1035200300 KingFisher 96 and consumables KingFisher 96 Magnetic Particle Processor 100 240V 50 60Hz 5400500 KingFi
12. ast a 16 bit color Monitor 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 24 InviMag SalivaGene DNA Kit KF96 0615 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 compatibility with various downstream applications Damaged DNA could perform poorly in applications such as genomic Southern blotting long template PCR Storage of DNA A working stock of DNA can be stored at 2 8 C for several weeks For long term storage the DNA should be stored at 20 C However storage at 20 C may cause shearing particularly if the DNA is exposed to repeated freeze thawing cycles Note that the used elution buffer 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 air dry than to use
13. ature 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 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 ocrew 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 KF96 0615 Scheme of the InviMag SalivaGene DNA Kit KF96 Please read protocols prior the start of the preparation carefully Important Prefill all plates with the appropriate b
14. ce for starting amounts is given in the individual protocols All protocols work with aliquots of the transport media in which fresh saliva samples are collected The DNA is stable for up to 12 months at room temperature in the transport media SalivaGene DNA Stabilizer The aliquots of the stabilization buffer may be processed on the same day as collection or stored for future processing For long term storage freeze the sample at 20 C STRATEC Molecular will be released of its responsibilities if other sample materials than described in the Intended Use are processed or if the sample preparation protocols are changed or modified Principle and procedure The InviMag SalivaGene DNA Kit KF96 procedure comprises following steps o collection of the material using a SalivaGene Collection Set lysis of cells in presence of non chaotropic conditions at elevated temperature in the Stabilization Buffer SalivaGene DNA Stabilizer protein digestion Proteinase K binding of the genomic DNA to magnetic beads MAP Solution B washing of the beads and elimination of contaminants and ethanol elution of pure genomic or bacterial DNA O O O O O This manual contains two protocols according to the different requirements of the starting materials and the extraction instrument used Lysis Samples are lysed in presence of non chaotropic conditions at elevated temperature in the Stabilization Buffer SalivaGene DNA Stabilizer Protein dig
15. ces 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 pipette tips between different liquid transfers To avoid cross contamination 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 Avoid 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 his kit should only be used by trained personnel O O O Preparing reagents and buffers Before starting a run equilibrate all reagents at room temperature Where necessary gently mix and redissolve any precipitates by warming up to 30 C Swirl gently to avoid foaming 1 Add the required amount of ddH5O to Proteinase K containing tube see below Vortex for 5 s Repeat this step for the Lysozyme 2 Add the required amount of ethanol to Wash Buffer and Wash Buffer Il and isopropanol to Binding Buffer A 3 Elution Buffer is ready to use 1x 96 DNA extractions Add 21 ml 99 7 Isopropanol to the Binding Buffer A Mix by intensive shaking by inverting for 1 min Shortly be
16. e provided Elution Plate as a Tip Plate 14 InviMag SalivaGene DNA Kit KF96 0615 Protocol 1 Human genomic 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 Important For working with the KingFisher instrument please carefully read the manufacturer s documents 1 Switch on the KF96 KFflex96 instrument 2 Prefill all Deep Well Plates as described below Note Please avoid evaporation of the prefilled buffer components by sealing the Deep Well Plates with a sealing foil or with parafilm Important Mix the bottle with the MAP B Solution by vigorously vortexing Binding Plate Washing plate 1 Washing plate 2 Washing plate 3 Elution Plate Carefully transfer 500 ul of the stabilized sample into the cavity of the Binding Plate 2 ml DWP prefilled with 20 ul Proteinase K Add 500 ul Wash Buffer I into the cavities of a 2 ml Deep Well Plate Add 600 ul Wash Buffer II into the cavities of a 2 ml Deep Well Plate Add 600 ul Wash Buffer Il into the cavities of a 2 ml Deep Well Plate Add 150 ul Elution Buffer into the cavities of the KF Elution Plate 3 Choose the program InviMag SalivaGene KFflex96 Human or InviMag SalivaGene KF96 Human and press the START button 4 Load the prefilled plates into the KingFisher instrument by following the specification
17. einase K and Lysozyme into aliquots and storage at 20 C is recommended Wash Buffers and Binding Buffer charged with either ethanol or isopropanol should be appropriate sealed and stored at RT The MAP Solution B magnetic beads should be stored at 2 8 C Before use make sure that all components have room temperature If there are any precipitates visible within the provided solutions dissolve them by carefully warming up to 30 C Quality control and product warranty STRATEC Molecular warrants the correct function of the InviMag SalivaGene DNA Kit KF96 for applications as described in this manual Purchaser must determine the suitability of the product for its particular use Should any product fail to perform the applications as described in 5 InviMag SalivaGene DNA Kit KF96 0615 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 KF96 have been tested separately against predetermined specifications routinely on lot to lot to ensure consistent product quality If you have any questions or problems regarding any aspects o
18. estion Proteins are 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 mixed with magnetic beads and the genomic DNA is bound to the beads Removing residual contaminations Contaminants are efficiently removed using Wash Buffer I and II while the genomic mitochondrial or bacterial DNA remains bound to the magnetic beads Elution of pure genomic DNA The nucleic acids are eluted from the magnetic beads using Elution Buffer and are ready to use in different downstream applications Yield and quality of DNA The amount of purified DNA in the InviMag SalivaGene DNA Kit KF96 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 downstream detection system The diagnostic tests should be performed accordingly to manufacturer s specifications 9 InviMag SalivaGene DNA Kit KF96 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 Servi
19. f InviMag SalivaGene DNA Kit KF96 or other STRATEC Molecular products please do not hesitate to contact us A copy of STRATEC Molecular s terms and conditions can be obtained upon request or are presented at the STRATEC Molecular webpage 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 KF96 is suitable for a reproducible purification of total DNA from up to 500 ul stabilization media of saliva or swab 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 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 f
20. fore use mix by inverting several times Add 1 1 ml ddH5O to each tube with Proteinase K mix thoroughly until completely dissolving and store the tubes at 20 C Add 1 1 ml ddH5O to each tube with Lysozyme mix thoroughly and store the tubes at 20 C Add 30 ml of 96 100 ethanol to the bottle Wash Buffer I mix thoroughly and store with tightly closed cap at RT Add 105 ml of 96 100 ethanol to each bottle Wash Buffer II mix thoroughly and store with tightly closed cap at RT 5x 96 DNA extractions Add 84 ml 99 796 Isopropanol to the Binding Buffer A Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times Add 10 5 ml ddH5O to the bottle with Proteinase K mix thoroughly until completely dissolving and store the tubes at 20 C We recommend making aliquots and storing them at 20 C Add 10 5 ml ddH5O to the bottle with Lysozyme mix thoroughly and store the tubes at 20 C We recommend making aliquots and storing them at 20 C Add 80 ml of 96 100 ethanol to each bottle Wash Buffer I mix thoroughly and store with tightly closed cap at RT Add 140 ml of 96 100 ethanol to each bottle Wash Buffer Il mix thoroughly and store with tightly closed cap at RT 10 InviMag SalivaGene DNA Kit KF96 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 plea
21. given on the display and confirm every step by pressing the button START When all prefilled plates are loaded into the KingFisher machine press the button START again to execute the assay file The run will last approximately 40 50 minutes After protein degradation the machine will be paused and 20 ul MAP Solution B and 200 ul Binding Buffer A have to be added to each sample containing cavity in the Binding Plate After addition of both reagents press the START button once again to continue with the run From here the machine will finish the purification process without any further user interaction 15 InviMag SalivaGene DNA Kit KF96 0615 Protocol 2 Bacterial 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 Important For working with the KingFisher instrument please carefully read the manufacturer s manual 5 Switch on the KF96 KFflex96 instrument 6 Prefill all Deep Well Plates as described below Note Please avoid evaporation of the prefilled buffer components by sealing the Deep Well Plates with a sealing foil or with parafilm Important Mix the bottle with the MAP B Solution by vigorously vortexing Binding Plate Carefully transfer 500 ul of the sample into the cavity of the Binding Plate 2 ml DWP prefilled with 20 ul Lysozyme Washing
22. ilization 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 oaliva 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 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 KF96 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 temper
23. indings 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 or from parasites nor for 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 is responsible to validate the performance of the STRATEC Molecular product for any particular use STRATEC Molecular does not provide for validation of performance characteristics of the product with respect to specific applications STRATEC Molecular products may be used e g in clinical diagnostic laboratory systems conditioned upon the complete diagnostic system of the laboratory the laboratory has been validated pursuant to CLIA 88 regulations in the U S or equivalents in other countries All products sold by STRATEC Molecular are subject to extensive quality control procedures according to ISO 9001 2000 and ISO EN 13485 and are warranted to perform as described herein Any problems incidents or defects shall be reported to STRATEC Molecular immediately upon detection thereof 6 InviMag SalivaGene DNA Kit KF96 0615 The chemicals and the plastic parts are for laboratory use only they must be stored in the labo
24. ixing Postmix Collect count Collect time s Washing Plate 3 Dry time Tip position Elution Plate Precollect Release time speed Mixing time speed Heating temperature C Preheat Postmix Collect count Collect time s Washing Plate 3 Release time speed Tip Plate 22 No 00 00 10 Fast 00 01 00 Fast om ZZ O Oo 00 03 30 Outside well tube No 00 00 10 Medium 00 10 00 Slow 60 No No 5 10 00 00 30 Fast InviMag SalivaGene DNA Kit KF96 0615 Troubleshooting low amount of DNA degraded or sheared DNA problems with subsequent applications e g in PCR As260 A280 ratio for purified DNA is low insufficient cell lysis due to decreased repeat the DNA purification procedure with a new sample anc Proteinase K activity insufficient lysis due to insufficient mixing with lysis buffer SalivaGene DNA Stabilizer low percentage alcohol used incomplete elution pH of water incorrect acidic incorrect storage of starting material Old material ethanol in the eluted DNA salt in the eluate reduced sensitivity of amplification reaction insufficient cell lysis and protein degradation due to decreased Proteinase K activity insufficient lysis due to insufficient mixing with lysis buffer freshly prepared Proteinase K stock solution Be sure to store the stock solution at 20 C repeat the DNA purification procedure with a new sample Be sure
25. l Lysis Plate Precollect Release beads Mixing time speed Heating temperature C Preheat Postmix Collect beads Lysis Plate Message Dispensing volume ul Name Volume ul Lysis Plate Precollect Release time speed Mixing time speed Heating during mixing Postmix Collect count Collect time s 18 No No 00 10 00 Medium 37 Yes No No Add Proteinase K 20 Proteinase K 20 No Yes 00 20 00 Medium 60 Yes No No Add Beads Buffer 220 Binding Buffer MAP B Solution 220 No 00 00 10 Fast 00 05 00 Medium InviMag SalivaGene DNA Kit KF96 0615 57 Washl Beginning of step Mixing heating End of step Wash2 Beginning of step Mixing heating End of step Wash3 Beginning of step Mixing heating End of step Dry Hlution Beginning of step Mixing heating End of step Bead Removal Leave Washing Plate 1 Precollect Release time speed Mixing time speed Heating during mixing Postmix Collect count Collect time s Washing Plate 2 Precollect Release time speed Mixing time speed Heating during mixing Postmix Collect count Collect time s Washing Plate 3 Precollect Release time speed Mixing time speed Heating during mixing Postmix Collect count Collect time s Washing Plate 3 Dry time Tip position Elution Plate Precollect Release time speed Mixing time speed Heating temperature
26. n either be transferred onto the corresponding workstation s or be started directly from within the Bindlt software after assay import Please keep in mind that assay s run from within the Bindlt software are not stored in the workstation memory Important Be advised that Bindlt SW 3 2 or higher versions use a new unique file extension Therefore it is not possible to import assay files created with Bindlt 3 2 or higher versions into older Bindlt software versions Please ask your local Thermo Scientific distributor for a software update Note When creating assay files for usage with KingFisher instruments in combination with Microtiter Deep Well plates e g Thermo Electron it is essential to use the KingFisher software 3 2 or higher versions for 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 AO pere ig MS Windows XP Pro with SP3 Windows Vista SP2 Windows 7 Disk space 500 MB free disk space Processor Intel Pentium 1 GHz Memory 1 GB RAM Serial ports available 1 for KFmL connection USB ports available 1 for KF96 KFflex96 KFDuo connection Mouse or equivalent is required XVGA monitor with at least 1024x768 resolution and at le
27. ng of step Mixing heating End of step Adjust Binding Reagent s Binding Beginning of step Mixing heating End of step Washl Beginning of step Mixing heating End of step Wash2 Beginning of step Mixing heating End of step 96 DW tip comb Tip Plate Lysis Plate Precollect Release beads Mixing time speed Heating temperature C Preheat Postmix Collect beads Lysis Plate Message Dispensing volume ul Name Volume ul Lysis Plate Precollect Release time speed Mixing time speed Heating during mixing Postmix Collect count Collect time s Washing Plate 1 Precollect Release time speed Mixing time speed Heating during mixing Postmix Collect count Collect time s Washing Plate 2 Precollect Release time speed Mixing time speed Heating during mixing Postmix Collect count Collect time s 21 No Yes 00 20 00 Medium 60 Yes No No Add Beads Buffer 720 Binding Buffer MAP B Solution 220 No 00 00 10 Fast 00 05 00 Medium 00 00 10 Fast 00 01 30 Fast Id om O o No 00 00 10 Fast 00 01 00 Fast cyt Mg z O o InviMag SalivaGene DNA Kit KF96 0615 3 Wash3 Beginning of step Mixing heating End of step Drying Elution Beginning of step Mixing heating End of step Bead Removal Leave Washing Plate 3 Precollect Release time speed Mixing time speed Heating during m
28. oved leaving the pure DNA to be eluted in Elution Buffer In case of extracting bacterial DNA an additional lysis step with Lysozyme is performed 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 The method requires only minimal user interaction allowing safe handling of potentially infectious samples The procedure is designed to avoid sample to sample cross contamination 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 RFLP AFLP analysis restriction enzyme digestion HLA typing pathogen detection O O O 0 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 PSP SalivaGene DNA Kit 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 KF96 0615 Sampling and sample storage Starting material The amount of starting material used in the InviMag SalivaGene DNA Kit KF96 procedure can vary slightly depending on the amount of DNA present in the sample Specific guidan
29. ratory and must not be used for purposes other than intended The product and its content are 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 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 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 infectious and be handled and discarded accordingly to local safety regulations European Community risks and safety phrases for the components of the InviMag SalivaGene DNA Kit KF96 to which they apply are listed below as follows Wash Buffer D warning H302 312 332 412 EUHO32 P273 Proteinase K SalivaGene DNA Stabilizer OD O danger danger H315 319 334 335 P280 305 351 338 310 405 H319 P305 351 338 H319 Causes serious
30. s with DNA purification The system combines the use of a prefilled SalivaGene Collection Tube for saliva or swab 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 40 50 min of high quality total DNA from up to 500 ul sample mixture The kit is designed for isolation of DNA from host organism as well as for isolation of DNA from pathogen microorganisms After collection the sample is transferred to the SalivaGene Collection Tube prefilled with SalivaGene DNA Stabilizer The SalivaGene DNA Stabilizer leads to an inactivation of DNases and prevents the degradation of the 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 for at least 12 months at ambient temperature The sample can alternatively be stored in this buffer at 20 C for 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 a PSP SalivaGene DNA isolation procedure At first the proteins are degraded with Proteinase K followed by binding of the DNA to magnetic beads while contaminants like potent inhibitors and proteins are rejected and rem
31. se consult the appropriate material safety data sheets MSDS These are available online in convenient and compact PDF format at www stratec com under each STRATEC Molecular kit and kit component Measuring cylinder 250 ml Disposable gloves Pipette and pipette tips Vortexer ddH O 96 100 ethanol lsopropanol O O O 0000 The InviMag SalivaGene DNA Kit KF96 is validated with 2 Propanol Rotipuran gt 99 7 p a ACS ISO Order no 6752 from Carl Roth Possible suppliers for Isopropanol 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 o Samples and buffers should be thoroughly mixed and should have room temperature o The elution can be done by using lower amount of Elution Buffer This may result in a higher concentration of DNA but pay attention that minimum volume for elution is 50 ul However this will lead to a reduced yield and we strongly recommend to never use a volume smaller than 100 ul o The volume of the eluted DNA may be lower than the added volume of Elution Buffer o The Elution Buffer doesn t contain EDTA o The yield can be increased if the incubation time with Elution Buffer will be prolonged 11 InviMag SalivaGene DNA Kit KF96 0615 PSP Treatment with SalivaGene Collection Module II Intended Use Collection of human saliva samples for DNA stab
32. sher 96 Head for Deep Well plate 24073430 KingFisher 96 tip comb for PCR magnets 8 x 10 pcs box 97002514 KingFisher 96 tip comb for KF magnets 10 x 10 pcs box 97002524 KingFisher 96 tip comb for DW magnets 10 x 10 pcs box 97002534 KingFisher 96 KF plate 200ul 48 plates box 97002540 Microtiter deep well 96 plate 50 plates box 95040450 Possible suppliers for Isopropanol 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 26 InviMag SalivaGene DNA Kit KF96 0615 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 1G3g02 06 2015
33. stratecee molecular 9 User manual InviMag SalivaGene DNA Kit KF96 for use on KingFisher 96 and KingFisher Flex Thermo Fisher Scientific for automated purification of total DNA from SalivaGene stabilized clinical swab mouth brushes amp saliva samples with magnetic beads 7435060X00 kaal STRATEC Molecular GmbH D 13125 Berlin v Instruction for InviMag SalivaGene DNA Kit KF96 The InviMag SalivaGene DNA Kit KF96 is used to stabilize and purify human and or bacterial genomic DNA from salivagene quantitatively by use of magnetic beads The InviMag SalivaGene DNA Kit KF96 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 months with a very efficient isolation method for high quality DNA The kit is neither suitable for the isolation of DNA from stool samples or 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
34. uffers and volumes as described below before starting a run Tip Plate Insert the KF96 Tip Comb for DW magnets on a Tip Plate Binding Plate Prepare the Binding plate as described below Washing Plate 1 Add 500 ul Wash Buffer I to a 2 0 ml Deep Well Plate Washing Plate 2 Add 600 ul Wash Buffer II to a 2 0 ml Deep Well Plate Washing Plate 3 Add 600 ul Wash Buffer II to a 2 0 ml Deep Well Plate Elution Plate Add 150 ul Elution Buffer to a KF Elution Plate same size as Tip Plate Bacteria DNA Transfer 500 ul stabilized saliva into the Lysis Plate add 20 ul of Lysozyme and start the program InviMag oalivaGene DNA Bacteria KFflex96 or InviMag SalivaGene DNA Bacteria KF96 After the first lysis step the user will be prompted to add 20 ul Proteinase K and confirm it by pressing Start Human DNA Transfer 500 ul of the stabilized sample into the Lysis Plate add 20 ul Proteinase K and start the program InviMag SalivaGene DNA Human KFflex96 InviMag SalivaGene DNA Human KF96 After the lysis step the user will be prompted to add 200 ul Binding Buffer A follow preparing instructions and 20 ul MAP Solution B during a pause DNA binds to magnetic particles Magnetic separation mm EE Washing of the particle fixed genomic DNA ie d Magnetic separation Elution of nucleic acids Magnetic Separation aci EK ET Pure DNA Elution Plates and Tip Plates are identically Use on
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