InviMag Bacteria DNA Mini Kit/ KFmL User manual
Contents
1. 1 5 ml Receiver Tubes 15 5x15 6 x 50 Manual Add 7 ml 99 7 Isopropanol to the Binding Buffer B6 Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times Add 7 5 ml of 96 100 ethanol to the bottle Wash Buffer I Add 42 ml of 96 100 ethanol to the bottle Wash Buffer Il Mix thoroughly and always keep the bottles firmly closed Add 21 ml 99 7 Isopropanol to each Binding Buffer B6 Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times Add 30 ml of 96 100 ethanol to each bottle Wash Buffer I Add 105 ml of 96 100 ethanol to the bottle Wash Buffer Il Mix thoroughly and always keep the bottles firmly closed Initial steps Add 56 ml 99 7 Isopropanol to each Binding Buffer B6 Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times Add 80 ml of 96 100 ethanol to each bottle Wash Buffer I Add 140 ml of 96 100 ethanol to each bottle Wash Buffer Il Mix thoroughly and always keep the bottles firmly closed Plastic to be supplied by us er see order information KingFisher ml Tip Combs 3 60 KingFisher ml Tube Strips 15 5x15 300 InviMag Bacteria DNA Mini Kit KFmL 0515 Symbols LOT Lot number Catalogue number El i 1 K Expiry date Consult operating instructions Temperature lim2. Binding Beginning of step Precollect No 19 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 Wash1 Plate Bacteria B Washing 1 Beginning of step Precollect No Release time hh mm ss 00 00 30 Release speed Medium Mixing pause parameters Pause for manual handling No Mixing time hh mm ss 00 02 00 Mixing speed Slow End of step Postmix No Collect count 3 Collect time s 2 Wash2 Plate Bacteria C Washing 2 Beginning of step Precollect No Release time hh mm ss 00 00 30 Release speed Medium Mixing pause parameters Pause for manual handling No Mixing time hh mm ss 00 01 00 Mixing speed Slow End of step Postmix No Collect count 3 Collect time s 2 Wash3 Plate Bacteria D Washing 3 Beginning of step Precollect No Release time hh mm ss 00 00 30 Release speed Medium Mixing pause parameters Pause for manual handling No Mixing time hh mm ss 00 01 00 Mixing speed Slow End of step Postmix No Collect count 3 Collect time s 2 InviMag Bacteria DNA Mini Kit KFmL 0515 Drying Plate Bacteria D Washing 3 Dry time hh mm ss 00 05 00 Tip position Outside well tube Elution Plate Bacteria E Elution Beginning of st
3. 15 extractions Add 7 ml 99 7 Isopropanol to the Binding Buffer B6 Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times Add 7 5 ml of 96 100 ethanol to the bottle Wash Buffer I Mix shortly and keep the bottle always firmly closed Add 42 ml of 96 100 ethanol to the bottle Wash Buffer II Mix shortly and keep the bottle always firmly closed 75 extractions Add 21 ml 99 7 Isopropanol to each Binding Buffer B6 Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times Add 30 ml of 96 100 ethanol to each bottle Wash Buffer I Mix shortly and keep the bottle always firmly closed Add 105 ml of 96 100 ethanol to the bottle Wash Buffer II Mix shortly and keep the bottle always firmly closed 300 extractions Add 56 ml 99 7 Isopropanol to each Binding Buffer B6 Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times Add 80 ml of 96 100 ethanol each the bottle Wash Buffer I Mix shortly and keep the bottle always firmly closed Add 140 ml of 96 100 ethanol to each bottle Wash Buffer Il Mix shortly and keep the bottle always firmly closed 11 InviMag Bacteria DNA Mini Kit KFmL 0515 Scheme of the InviMag Bacteria DNA Mini Kit KFmL Please read protocols prior the start of the preparation carefully Please prepare the sample preparation outside the KingFishe
4. 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 24 InviMag Bacteria DNA Mini Kit KFmL 0515 stratecee molecular STRATEC Molecular GMbH Robert R ssle Str 10 13125 Berlin Germany Phone 49 30 94 89 29 01 Fax 49 30 94 89 29 09 E mail info berlin stratec com www stratec com 1G3101 05 2015
5. 20 C avoid thawing and freezing of the material old material often contains degraded DNA DNA does not perform well in downstream applications e g real time PCR or PCR ethanol carryover during elution salt carryover during elution increase drying time for removing of ethanol check up the Wash Buffers for salt precipitates If there are any precipitates solve these precipitates by careful warming ensure that the Wash Buffers are at room temperature low A260 A280 ratio from UV measurement eluted DNA is brown colored small part of the magnetic particles are left in the elution centrifuge down at full speed for 1 min and transfer supernatant to a new tube 21 InviMag Bacteria DNA Mini Kit KFmL 0515 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 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
6. 95 C for 10 min continuously shaking increases the lysis efficiency DNA Binding After lysis transfer app 450 ul of the lysed sample into the Tube A of the KingFisher tube and add 400 ul of Binding Buffer B6 and 20 ul MAP Solution A see also below Note Vortex the tube MAP Solution A vigorously before use Start the program InviMag Bacteria KFmL see instructions on page 17 13 InviMag Bacteria DNA Mini Kit KFmL 0515 Protocol 3 Isolation of DNA from bacteria pellets 1 x 10 bacteria cells Please read the instructions carefully and conduct the prepared procedure Attention Please be aware that you have to prepare the Binding Buffer B6 see instruction page 11 Lysis at 65 C for 10 min in a thermomixer Pellet the bacteria by centrifugation Resuspend the bacterial pelltet in 400 ul Resuspension Buffer R Transfer the resuspended sample into the Extraction Tube and vortex for 5 s Incubate the sample in a thermomixer at 65 C for 10 min continuously shaking increases the lysis efficiency During lysis see Preliminary Steps to process the sample onto the KingFisher System page 10 and follow the instructions Lysis at 95 C for 10 min in a thermomixer Place the Extraction Tube with the lysis mixture into a thermomixer and incubate at 95 C for 10 min continuously shaking increases the lysis efficiency DNA Binding After lysis transfer app 450 ul of the lysed sample into the Tube A of the Kin
7. ACS ISO Order no A3928 Order no 59304 1L F Order no 6752 10 InviMag Bacteria DNA Mini Kit KFmL 0515 Important notes Important points before starting a protocol Immediately upon receipt of the product inspect the product and its components as well as the package for any apparent damages 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 O OO 0O 0 Always change pipet tips between liquid transfers To avoid cross contamination we recommend the use of aerosol barrier pipet tips All centrifugation steps should be carried out at room temperature When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles Discard gloves if they become contaminated 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 air flow until the samples are lysed This kit should only be used by trained personnel Preparing reagents and buffers
8. dicarded Lysis Samples are lysed at non chaotropic conditions with different elevated temperatures while continuously shaking Lysis is performed in the presence of Lysozyme to break the cell wall of the bacteria Lysis Buffer to lyse the cells and Proteinase K to digest the proteins Every component is provided prefilled in the Extraction Tube Unlysed sample parts should be removed before the binding step Binding genomic DNA By adding Binding Buffer B6 and the MAP Solution A to the lysate optimal binding conditions are adjusted The DNA is adsorbed to the InviMag beads as the beads are mixed carefully by the magnetic rods Removing residual contaminants Contaminants are efficiently removed using two different Wash Buffers while the bacterial genomic DNA remains bound to the beads Elution of pure genomic DNA Genomic DNA is eluted from the beads using 100 ul Elution Buffer The eluted DNA is ready to use in different downstream applications Equipment and reagents to be supplied by user o Eppendorf Thermomixer o ddH O0 o Measuring cylinder 250 ml o optional octane o Disposable gloves o lsopropanol o Pipet with tips o 96 100 ethanol The InviMag Bacteria DNA Mini 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 pelea Applichem Sigma aie gt 2 Propanol f r die Molekularbiologie 2 Propanol Rotipuran gt 99 7 p a
9. license to practice PCR under any patents held by Hoffmann LaRoche Inc 8 InviMag Bacteria DNA Mini Kit KFmL 0515 Principle and procedure The InviMag Bacteria DNA Mini Kit KFmL procedure comprises following steps o lysis of sample material o binding the genomic DNA to the magnetic beads o washing the beads and elimination of waste and ethanol o elution of total DNA This manual contains 9 protocols The samples are lysed in an optimized buffer and enzyme mixture The lysates are transferred to the subsequent automatic purification procedure based on magnetic beads The DNA binds to magnetic particles followed by washing steps and the final elution The purified high quality DNA is ready to use for subsequent downstream applications like PCR amplification quantitative PCR real time PCR or other routine laboratory methods Sampling and storage of starting material Pathogens Lysteria ssp in food material For the detection of bacteria Lysteria in foods an enriched and cultivation following the EU regulations and 35 of the food law ios required An aliquot of the culture is used and the bacteria will be centrifuged after cultivation Best results are obtained with fresh material or material that has been immediately frozen and stored at 20 C or 80 C Repeated freezing and thawing cycles of stored samples should be avoided because this may lead to degraded DNA Bacterial cultures Bacterial cultures grow in the prese
10. 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 Puppene operating 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 Monitor color settings 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 22 InviMag Bacteria DNA Mini Kit KFmL 0515 General notes on handling DNA Nature of DNA The length and delicate physical nature of DNA require 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 n
11. al tips that have wide openings designed for pipetting genomic DNA Regular pipette tips pose no problem for plasmid DNA and other small 23 InviMag Bacteria DNA Mini Kit KFmL 0515 Ordering information Product Package size Catalogue No InviMag Bacteria DNA Mini Kit KFmL 15 extractions 2433150100 InviMag Bacteria DNA Mini Kit KFmL 75 extractions 2433150200 InviMag Bacteria DNA Mini Kit KFmL 300 extractions 2433150400 InviMag Bacteria DNA Mini Kit KFmL wp 15 extractions 2433150150 InviMag Bacteria DNA Mini Kit KFmL wp 75 extractions 2433150250 InviMag Bacteria DNA Mini Kit KFmL wp 300 extractions 2433150450 Single components Extraction Tube 10 tubes 7433301500 MAP Solution A 1 ml 7433305100 Resuspension Buffer R 30 ml 7433302200 Binding Buffer B6 30 ml 7433302100 Wash Buffer 30ml 7433303500 Wash Buffer II 18 ml 7433303600 Elution Buffer 15 ml 7433304000 KingFisher ml Tip Comb 1 piece 0030014400 KingFisher ml Tube 1 piece 0030014500 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 Carl Roth 2 Propanol 3 Applichem Sigma Rotipuran
12. al volume 2 x 60 ml final volume 2 x 160 ml 18 ml 45 ml 3 x 60 ml Wash Buffer II final volume 60 ml final volume 150 ml final volume 3 x 200 ml 1 5 ml Receiver Tubes 15 5x15 6 x 50 KingFisher ml Tip Combs 3 15 60 KingFisher ml Tube Strips 15 5x15 300 Manual 1 1 1 Initial steps Add 56 ml 99 7 Isopropanol to each Binding Buffer B6 Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times Add 80 ml of 96 100 ethanol to each bottle Wash Buffer I Add 140 ml of 96 100 ethanol to each bottle Wash Buffer Il Mix thoroughly and always keep the bottles firmly closed InviMag Bacteria DNA Mini Kit KFmL 0515 Kit contents of InviMag Bacteria DNA Mini Kit KFmL w o plastic Store the MAP Solution A at 2 8 Store diluted Proteinase K at 20 C Store all other kit components at room temperature 15 extractions 75 extractions 300 extractions Binding Buffer B6 final volume 10 ml final volume 2 x 30 ml Catalogue No 2433150150 2433150250 2433150450 Extraction Tube 15 75 6 x 50 Resuspension Buffer R 15 ml 60 ml 150 ml MAP Solution A 0 5 ml 2ximl 7 mi 3 ml 2x9 ml 2x 24 ml final volume 2 x 80 ml Wash Buffer Il final volume 60 ml final volume 150 ml Elution Buffer 2 ml 15 ml 60 ml 7 5 ml 2 x 30 ml 2 x 80 ml west Buter final volume 15 ml final volume 2 x 60 ml final volume 2 x 160 ml 18 ml 45 ml 3x60 ml final volume 3 x 200 ml
13. ance 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 Bacteria DNA Mini 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 Bacteria DNA Mini 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 5 InviMag Bacteria DNA Mini Kit KFmL 0515 Intended use The InviMag Bacteria DNA Mini Kit KFmL allows for rapid and economic isolation of high quality bacterial DNA from cell free body fluids paper points bacterial species tissue and food samples paraffin embedded tissue blood urine swabs and water using the InviMag technology This kit technology yields genomic DNA from bacteria from different human sources that is free of proteins nucleases and other impurities and is ready to use for different downstream applications like PCR quantitative PCR real time PCR or other routine methods For reproducible and high yields
14. appropriate sample storage is essential The purified DNA is of high quality 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 cultured or isolated cells from dried blood stains nor from stool samples fungi parasites or the purification of RNA The application of the kit for isolation and purification of viral DNA has not been evaluated The included chemicals are only useable once Differing 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 spe
15. aterial also leads to reduced length and yield of purified DNA The thawing process should be performed in the Extraction Tube The amount of purified DNA from max 10 mg tissue sample depends on the nature of starting material 9 InviMag Bacteria DNA Mini Kit KFmL 0515 Urine The bacteria must be centrifuged and the supernatant completely removed urea contaminations can inhibit PCR reactions Best results are obtained with fresh pellets or frozen pellets Repeated freezing and thawing cycles of stored samples should be avoided because this may lead to degraded DNA The amount of purified DNA from max 15 50 ml urine depends on the bacterial titer Swabs saliva The protocol works with fresh saliva prepared swabs as well as with dried swabs Please note that DNA from stored and dried swabs are often characterized by isolation of apoptotic DNA visible on agarose gel as typical apoptotic DNA banding pattern The protocol has not been validated for isolation of DNA from swabs which are stored in special storage buffers from other providers 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 Sample preparation Bacteria must be cultivated at special conditions An aliquot of the bacterial suspension is used to get a bacterial pellet by centrifugation at high speed for 5 min The supernatant is
16. c DNA especially from gram positive bacteria of different sources followed by binding of the bacterial nucleic acids to the InviMag Beads washing steps and final elution High extraction efficiency and detection sensitivities are realized The hands on time necessary for the whole procedure is reduced to a minimum The kit technology yields bacterial DNA from different human samples that is free of proteins nucleases and other impurities and ready to use for downstream application like PCR quantitative PCR real time PCR or other routine laboratory methods The procedures require minimal interaction by the user allowing safe handling of potentially infectious samples The procedures are optimized to avoid sample to sample_ cross contamination No toxic or hazardous chemicals like phenol chloroform or 8 Mercaptoethanol are used Traditional time killing procedures can be replaced using the InviMag Bacteria DNA Mini Kit KFmL Due to the high purity the isolated total 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 quantitative PCR like TaqMan und LightCycler technology Mircoarray application RFLP Analysis O O O O 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 Bacteria DNA Mini Kit KFmL cannot be construed as an authorization or implicit
17. cific 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 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 6 InviMag Bacteria DNA Mini Kit KFmL 0515 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 teste
18. d the liquid waste generated by the InviMag Bacteria DNA Mini 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 Bacteria DNA Mini Kit KFmL to which they apply are listed below as follows Wash Buffer I contains a chaotropic salt which is an irritant Wash Buffer Proteinase K included in the Extraction Tube warning danger H302 312 332 412 EUH032 P273 H315 319 334 335 P280 305 351 338 310 405 H319 Causes serious 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 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 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 St
19. ecessary to ensure it will function 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 nitrogen and stored at 80 C This procedure minimizes degradation of crude DNA by limiting the activity of endogenous nucleases Storage of DNA Store genomic DNA at 2 8 C Storing genomic DNA at 20 C may cause shearing of DNA particularly if the DNA is exposed to repeated freeze thaw cycles Plasmid DNA and other small circular DNA can be stored at 2 8 C or at 20 C Drying dissolving and pipetting DNA Avoid overdrying genomic DNA after ethanol precipitation It is better to let it air dry than to use a vacuum although vacuum drying can be used with caution Plasmid DNA and other small circular DNAs can be vacuum dried To help dissolve the DNA carefully invert the tubes several times after adding buffer and tap the tube gently on the side Alternatively let the DNA stand in buffer overnight at 2 8 C Minimize vortexing of genomic DNA because this may cause shearing Avoid vigorous pipetting Pipetting genomic DNA through small tip openings causes shearing or nicking One way to decrease shearing of genomic DNA is to use speci
20. ek N 13 13 14 14 15 15 16 16 17 17 21 22 23 24 2 InviMag Bacteria DNA Mini Kit KFmL 0515 Kit contents of InviMag Bacteria DNA Mini Kit KFmL Store the MAP Solution A at 2 8 C Store diluted Proteinase K at 20 C Store all other kit components at room temperature 15 extractions 75 extractions 300 extractions Binding Buffer B6 final volume 10 ml final volume 2 x 30 ml Catalogue No 2433150100 2433150200 2433150400 Extraction Tube 15 75 6 x 50 Resuspension Buffer R 15 ml 60 ml 150 ml MAP Solution A 0 5 ml 2x1ml 7 mi 3 ml 2x9 ml 2 x 24 ml final volume 2 x 80 ml Add 7 ml 99 7 Isopropanol to the Binding Buffer B6 Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times Add 7 5 ml of 96 100 ethanol to the bottle Wash Buffer I Add 42 ml of 96 100 ethanol to the bottle Wash Buffer Il Mix thoroughly and always keep the bottles firmly closed Add 21 ml 99 7 Isopropanol to each Binding Buffer B6 Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times Add 30 ml of 96 100 ethanol to each bottle Wash Buffer I Add 105 ml of 96 100 ethanol to the bottle Wash Buffer Il Mix thoroughly and always keep the bottles firmly closed Elution Buffer 2ml 15ml 60 ml 7 5 ml 2x 30 ml 2x 80 ml Wash Buffer final volume 15 ml fin
21. ep Precollect No Release time hh mm ss 00 00 30 Release speed Medium Mixing pause parameters Pause for manual handling No 20 Mixing time hh mm ss 00 05 00 Mixing speed Slow End of step Postmix No Collect count 4 Collect time s 3 Bead Removal Plate Bacteria D Washing 3 Release time hh mm ss 00 00 30 Release speed Fast InviMag Bacteria DNA Mini Kit KFmL 0515 Troubleshooting Problem Probable cause Comments and suggestions low amount of extracted DNA low concentration of extracted DNA degraded or sheared DNA insufficient lysis incomplete elution low amount of MAP Solution A too much Elution Buffer incorrect storage of starting material incorrect storage of starting material old material increase lyses time but prevent too long lyses tome because this also decrease yield reduce amount of starting material take higher volume of Elution Buffer be sure you pipet the Elution Buffer with the right amount to the right position mix MAP Solution A thoroughly before pipetting to the KingFisher tube elute the DNA with lower volume of Elution Buffer ensure that the storage of starting material was correctly avoid thawing of the material ensure that the storage of starting material was correctly avoid thawing of the material ensure that the starting material is fresh or stored under appropriate condition for long time storage at
22. eserved 1 InviMag Bacteria DNA Mini Kit KFmL 0515 Content Kit contents of InviMag Bacteria DNA Mini Kit KFmL Kit contents of InviMag Bacteria DNA Mini Kit KFmL w o plastic Symbols Storage Quality control Intended use Product use limitation Safety information Product characteristic of the InviMag Bacteria DNA Mini Kit KFmL Principle and procedure Sample preparation Equipment and reagents to be supplied by user Important notes Preparing reagents and buffers Scheme of the InviMag Bacteria DNA Mini Kit KFmL Protocol 1 Isolation of bacteria from cell free body fluids serum plasma cerebrospinal fluid liquor Protocol 2 Isolation of DNA from periodonthopathogenic bacteria from paper points Protocol 3 Isolation of DNA from bacteria pellets 1 x 10 bacteria cells Protocol 4 Isolation of DNA from swabs or 200 ul rinsed liquid from swabs Protocol 5 Isolation of bacterial DNA from tissue biopsies Protocol 6 Isolation of bacterial DNA from paraffin embedded tissue Protocol 7 Isolation of bacterial DNA from urine samples Protocol 8 Isolation of bacterial DNA from blood samples Protocol 9 Isolation of bacterial DNA from water samples no waste water Preliminary steps to process the sample onto the KingFisher system For self programming of the KFml system program InviMag Bacteria KFmL Troubleshooting Appendix General notes on handling DNA Ordering information OO MW N OD DHD A aA a fF WwW ek ek
23. gFisher tube and add 400 ul Binding Buffer B6 and 20 ul MAP Solution A see also below Note Vortex the tube MAP Solution A vigorously before use Start the program see instructions on page 17 Protocol 4 Isolation of DNA from swabs or 200 ul rinsed liquid from swabs Please read the instructions carefully and conduct the prepared procedure Attention Please be aware that you have to prepare the Binding Buffer B6 see instruction page 11 Lysis at 65 C for 10 min in a thermomixer Place the swab into the Extraction Tube and add 400 ul of Resuspension Buffer R Vortex for 5s Place the Extraction Tube tube in a thermomixer and incubate while continuously shaking for 15 min at 65 C During lysis see Preliminary Steps to process the sample onto the KingFisher System page 17 and follow the instructions Lysis at 95 C for 10 min in a thermomixer Place the Extraction Tube with the lysis mixture in a thermomixer and incubate at 95 C for 10 min continuously shaking increases the lysis efficiency DNA Binding After lysis transfer app 450 ul of the lysed sample into the Tube A of the KingFisher tube and add 400 ul of Binding Buffer B6 and 20 ul MAP Solution A see also below Note Vortex the tube MAP Solution A vigorously before use Start the program see instructions on page 17 Important Note To get the maximum yield of bacterial DNA it is essential to keep the swab inside the reaction tube during lysis It is p
24. he lysis procedure During lysis see under Preliminary Steps to process the sample onto the KingFisher System page 17 and follow the instructions Lysis at 95 C for 10 min in a thermomixer Place the Extraction Tube with the lysis mixture into a thermomixer and incubate at 95 C for 10 min continuously shaking increases the lysis efficiency DNA Binding After lysis transfer approximately 450 ul of the lysed sample into the Tube A of the KingFisher tube and add 400 ul Binding Buffer B6 and 20 ul MAP Solution A see also below Note Vortex the tube MAP Solution A vigorously before use Start the program see instructions on page 17 Protocol 2 Isolation of DNA from periodonthopathogenic bacteria from paper points Please read the instructions carefully and conduct the prepared procedure Attention Please be aware that you have to prepare the Binding Buffer B6 see instruction page 11 Lysis at 65 C for 10 min in a thermomixer Transfer the paper point into the Extraction Tube and add 400 ul Resuspension Buffer R to the Extraction Tube Vortex for 5 s Incubate the sample in a thermomixer at 65 C for 10 min continuously shaking increases the lysis procedure During lysis see Preliminary Steps to process the sample onto the KingFisher System page 17 and follow the instructions Lysis at 95 C for 10 min in a thermomixer Place the Extraction Tube with the lysis mixture into a thermomixer and incubate at
25. he supernatant into the Tube A of the KingFisher tube and add 400 ul of Binding Buffer B6 and 20 ul MAP Solution A see also below Note Vortex the tube MAP Solution A vigorously before use Start the program see instructions on page 17 Protocol 6 Isolation of bacterial DNA from paraffin embedded tissue Please read the instructions carefully and conduct the prepared procedure Attention Please be aware that you have to prepare the Binding Buffer B6 see instruction page 11 Deparaffination Transfer the sample into a 1 5 ml reaction tube Add 1 ml octane and vortex carefully to dissolve the paraffin Follow the dissolution until the tissue sample looks transparent while paraffin is still white Centrifuge for 2 min at maximum speed to collect the tissue sample Discard the supernatant very careful This step should be repeated if paraffin remains are still present in the sample Add 0 5 ml 96 ethanol to the tissue sample and mix the tube thoroughly Centrifuge shortly and remove the ethanol by aspiration with a pipette Then incubate the open tube at 56 C to evaporate residual ethanol Lysis at 65 C for 30 60 min in a thermomixer Transfer the deparaffinized tissue sample into the Extraction Tube and add 400 ul Resuspension Buffer R Close the cap and vortex for 5 s Place the Extraction Tube in a thermomixer and incubate for 30 60 min at 65 C while continuously shaking Lysis time can be increased if the lysis is incomple
26. in in a thermomixer Add 400 ul Resuspension Buffer R to the sediment and resuspend the pellet by pipetting up and down Transfer the resuspended sample into the Extraction Tube close the cap and vortex for 5 s Incubate the sample in a thermomixer at 65 C for 10 min continuously shaking increases the lysis efficiency During lysis see Preliminary Steps to process the sample onto the KingFisher System page 17 and follow the instructions Lysis at 95 C for 10 min in a thermomixer Place the Extraction Tube in a thermomixer and incubate at 95 C for 10 min continuous shaking increases the lysis efficiency DNA Binding After lysis transfer app 450 ul of the lysed sample into the Tube A of the KingFisher tube and add 400 ul Binding Buffer B6 and 20 ul MAP Solution A Note Vortex the tube MAP Solution A vigorously before use 5 Start the program see instructions on page 17 Preliminary steps to process the sample onto the KingFisher system 1 During sample lysis prefill the tubes of the KingFisher tube strips with the required buffers and appropriate volumes KingFisher ml Tube Strip Setup Tube A 400 ul Binding Buffer B6 and 20 ul MAP Solution A Note It is important to mix the bottle with MAP Solution A carefully by vigorously shaking or vortexing Tube B 800 ul Wash Buffer Tube C 800 ul Wash Buffer II Tube D 800 ul Wash Buffer II Tube E 200 ul Elution Buffer 2 Insert the filled KingFisher tube strips into the K
27. ingFisher System on the right position 3 Insert the KingFisher tip comb into the instrument 17 InviMag Bacteria DNA Mini Kit KFmL 0515 The following extraction steps run automatically on the KingFisher system Start the program InviMag Bacteria KFmL Important Notes 1 After finishing the extraction protocol the Tube E contains the extracted DNA Store the DNA under adequate conditions We recommend transferring the extracted DNA into 1 5 ml receiver tubes for further storage and the freeze the DNA at 20 C 2 If the extracted DNA contains carryover of magnetic particles transfer the DNA into a 1 5 ml reaction tube and centrifuge at maximum speed for 1 min and transfer the DNA containing supernatant into a new tube Automatic extraction steps running on the KingFisher system 1 Binding of the DNA Automatically sample mixing for 5 min MAP separation Moving of the MAP into the Tube B 2 First Washing Automatically sample mixing for 2 min MAP separation Moving of the MAP into the Tube C 3 Second Washing Automatically sample mixing for 1 min MAP separation Moving of the MAP into the Tube D 4 Third Washing and Drying Automatically sample mixing for 1 min MAP separation Drying the MAP outsight the Tube for 5 min Moving of the MAP into the tube E 5 Elution of the DNA Incubation of the MAP into the Tube E for 5 minutes by mixing MAP separation The MAP will than automatically removed into the well D d
28. isposal The extracted DNA can now be transferred into 1 5 ml receiver tubes Optional Carryover of magnetic particles should be removed by centrifugation at max speed for 1 min Transfer the cleared eluate into a new tube 18 InviMag Bacteria DNA Mini Kit KFmL 0515 For self programming of the KingFisher ml system program InviMag Bacteria KFmL Protocol Properties Name InviMag Universal Bacteria Kit KFmL Protocol template version 3 1 Instrument type KFmL Description KFmL protocol for isolation of bacterial DNA from liquid samples with the InviMag Bacteria DNA Kit KFmL Layout Data A Binding Plate type KingFisher tubestrip 1000 ul Reagents Name Lysed sample Volume yl 450 Type Sample Name Binding Buffer B6 Volume pl 200 Type Reagent Name MAP Solution A Volume pl 20 Type Reagent B Washing 1 Plate type KingFisher tub strip 1000ul Reagents Name Wash Buffer Volume ul 600 Type Reagent C Washing 2 Plate type KingFisher tub strip 1000ul Reagents Name Wash Buffer II Volume ul 800 Type Reagent D Washing 3 Plate type KingFisher tub strip 1000ul Reagents Name Wash Buffer II Volume ul 800 Type Reagent E Elution Plate type KingFisher tub strip 1000ul Reagents Name Elution Buffer Volume pl 200 Type Reagent Steps Data Tip1 Tip KingFisher ml tip comb Binding Plate Bacteria A
29. itation Q Do not reuse Storage All buffers and kit contents of the InviMag Bacteria DNA Mini Kit KFmL except MAP Solution A are stable for at least 12 months MAP Solution A is stable for at least 6 months All buffers and kit contents of InviMag Bacteria DNA Mini Kit KFmL except dissolved Proteinase K and MAP Solution A should be stored at room temperature Proteinase K Dissolved Proteinase K must be stored at 20 C Dividing the Proteinase K into aliquots and storage at 20 C is recommended MAP Solution A should be stored at 2 8 C Wash Buffer charged with ethanol should be stored at room temperature and should be appropriate sealed If there are any precipitates within the provided solutions solve these precipitates by careful warming up to room temperature up to 30 C Room temperature RT is defined as range from 15 30 C Quality control and product warranty STRATEC Molecular warrants the correct function of the InviMag Bacteria DNA Mini 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 perform
30. nce of a selective agent such as an antibiotic The yield and quality of the DNA depends on factors such host strain inoculation antibiotic and type of culture medium The bacteria will be centrifuged after cultivation Best results are obtained with fresh material or material that has been immediately frozen and stored at 20 C or 80 C Repeated freezing and thawing cycles of stored samples should be avoided because this may lead to degraded DNA Paper points Paper points can be stored at room temperature for up to 6 hours For long term storage we recommend dry storage at 2 8 C Blood Blood samples can be stored at room temperature for up to 6 hours or at 2 8 C for up to 24 hours For long term storage we recommend freezing samples at 20 C or 80 C Multiple thawing and freezing cyckes before isolating the DNA should be avoided If cryoprecipitates formed during thawing of frozen samples are visible avoid aspirating them The amount of purified DNA from max 100 ul whole blood depends on the white blood cell content of each blood sample Various different primary tubes and anticoagulants except heparin can be used to collect blood samples Tissue biopsy material Best results are obtained with fresh material or material that has been immediately frozen and stored at 20 C or 80 C Repeated freezing and thawing cycles of stored samples should be avoided because this may lead to degraded DNA Use of poor quality starting m
31. nd incubate for 15 min at 65 C while continuously shaking During lysis see Preliminary Steps to process the sample onto the KingFisher System page 17 and follow the instructions Lysis at 95 C for 10 min in a Thermomixer Place the Extraction Tube into a Thermomixer and incubate at 95 C for 10 min continuously shaking increases the lysis efficiency DNA Binding After lysis transfer app 450 ul of the lysed sample into the Tube A of the KingFisher tube and add 400 ul of Binding Buffer B6 and 20 ul MAP Solution A see also below Note Vortex the tube MAP Solution A vigorously before use Start the program see instructions on page 17 16 InviMag Bacteria DNA Mini Kit KFmL 0515 Protocol 9 Isolation of bacterial DNA from water samples no waste water Please read the instructions carefully and conduct the prepared procedure Attention Please be aware that you have to prepare the Binding Buffer B6 see instruction page 11 Attention Please STRATEC Molecular offers on request also a very special protocol for isolation of DNA from Legionella species from 1 L water samples Sample Collection Concentrate the starting sample e g 1 L by standard procedures like centrifugation or filtration Finally spin down the bacteria by centrifugation Decant the supernatant carefully and resuspend the sediment in 10 ml of 1x PBS Centrifugation for 5 min at 3500 rom Decant the supernatant carefully Lysis at 65 C for 10 m
32. nd the pellet in 400 ul Resuspension Buffer R Transfer the resuspended cells into the Extraction Tube and vortex for 5 s Incubate the sample in a thermomixer at 65 C for 10 min continuously shaking increases the lysis efficiency During lysis see Preliminary Steps to process the sample onto the KingFisher System page 17 and follow the instructions Lysis at 95 C for 10 min in a thermomixer Place the Extraction Tube with the lysis mixture in a thermomixer and incubate at 95 C for 10 min continuously shaking increases the lysis efficiency DNA Binding After lysis transfer app 450 ul of the lysed sample into the Tube A of the KingFisher tube and add 400 ul of Binding Buffer B6 and 20 ul MAP Solution A Note Vortex the tube MAP Solution A vigorously before use 5 Start the program see instructions on page 17 Protocol 8 Isolation of bacterial DNA from blood samples Please read the instructions carefully and conduct the prepared procedure Attention Please be aware that you have to prepare the Binding Buffer B6 see instruction page 11 Lysis at 65 C for 20 min in a thermomixer Mix up to 100 ul of whole blood with 300 ul Resuspension Buffer R If the starting volume of whole blood is lower than 100 ul add more Resuspension Buffer R The final volume schould be 400 ul Transfer the sample completely into the Extraction Tube and close the cap Vortex for 5 s Place the Extraction Tube in a thermomixer a
33. ore 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 Bacteria DNA Mini Kit KFmL 0515 Product characteristic of the InviMag Bacteria DNA Mini Kit KFmL max 10 bacteria depends on the approx 20 25 min Azeo A 280 1 10 mg tissue biopsy material starting material after lysis paraffin embedded tissue 1 100 ul whole blood 200 ul cell free body fluid 15 50 ml urine paper points swabs up to 1 water The InviMag Bacteria DNA Mini Kit KFmL allows for rapid and economic isolation of high quality genomic DNA from a broad range of bacteria species up to 10 bacteria cells and different sources using the RTP technology and a KingFisher ml workstation For the wide range of different samples the InviMag Bacteria DNA Mini Kit KFmL includes protocols adapted to the need of every kind of starting material All types of samples are transferred into the Extraction Tube together with a specially designed Resuspension Buffer R The prefilled buffer and enzymes lyse the samples stabilize nucleic acid and enhance the selective DNA adsorption to the beads Mostly the lysis is carried out at two different incubation temperatures in the Extraction Tube to increase the sensitivity In addition to the rigorous lysis procedure simple pretreating steps have been introduced ideally for purification of genomi
34. ossible to cut the shaft of the swab so that you the cap of the Extraction Tube can be closed It is also possible to perform the lysis step with an opened cap The removing of the swab from the Extraction Tube ahead of time will be lead to a dramatically reduced final yield After lysis carefully squeeze out the swab inside the tube wall and discard the swab 14 InviMag Bacteria DNA Mini Kit KFmL 0515 Protocol 5 Isolation of bacterial DNA from tissue biopsies Please read the instructions carefully and conduct the prepared procedure Attention Please be aware that you have to prepare the Binding Buffer B6 see instruction page 11 Lysis at 65 C for 30 60 min in a thermomixer Transfer 1 10 mg of the biopsy material into the Extraction Tube and add 400 ul Resuspension Buffer R Close the cap and vortex for 5 s Place the Extraction Tube in a thermomixer and incubate for 30 60 minutes at 65 C while continuously shaking Lysis time can be increased if the lysis step is not complete During lysis see Preliminary Steps to process the sample onto the KingFisher System page 17 and follow the instructions Lysis at 95 C for 10 min in a Thermomixer Place the Extraction Tube into a Thermomixer and incubate at 95 C for 10 min continuous shaking increases the lysis efficiency After lysis centrifuge the sample at max speed for 1 min to spin down unlysed material DNA Binding After centrifugation transfer app 450 ul of t
35. r mL platform following the instructions During lysis prefill the KingFisher Tube strips with required buffers and appropriate volumes Tube A 400 ul Binding Buffer B6 and 20 ul MAP Solution A Tube B 800 ul Wash Buffer Tube C 800 ul Wash Buffer Il Tube D 800 ul Wash Buffer Il Tube E 200 ul Elution Buffer Lysis of starting material in the Extraction Tube Add required amount of Resuspension Buffer R to the sample and transfer the sample to the Extraction Tube Vortex for 5 s Incubation in a Thermomixer at 65 C for 10 min continuously shaking increases the lysis efficiency Transfer of lysate to InviMag beads and Binding Buffer B6 follow preparing instructions in the 5 tube strip format DNA binding to the magnetic beads Magnetic separation Washing Magnetic separation Elution and magnetic separation Pure DNA 12 InviMag Bacteria DNA Mini Kit KFmL 0515 Protocol 1 Isolation of bacteria from cell free body fluids serum plasma cerebrospinal fluid liquor Please read the instructions carefully and conduct the prepared procedure Attention Please be aware that you have to prepare the Binding Buffer B6 see instruction page 11 Lysis at 65 C for 10 min in a thermomixer Mix 200 ul Resuspension Buffer R with 200 ul of liquid sample transfer the sample to the Extraction Tube and vortex for 5 s Incubate the sample in a thermomixer at 65 C for 10 min continuous shaking increases t
36. stratecee molecular User manual InviMag Bacteria DNA Mini Kit KFmL for use on KingFisher mL Thermo Fisher Scientific for automated purification of bacterial DNA from different types of specimen swabs tissue food paraffin embedded tissue urine blood or water samples with magnetic beads 2433150X00 taal STRATEC Molecular GmbH D 13125 Berlin Instruction InviMag Bacteria DNA Mini Kit KFmL The InviMag Bacteria DNA Mini Kit KFmL allows for rapid and economic isolation of high chromosomal bacterial DNA from cell free body fluids cerebrospinal fluid liquor urine paper points bacterial pellets tissue samples paraffin embedded tissue blood urine swabs and water using the InviMag technology The kit is neither validated for the isolation of genomic DNA from cultured or isolated cells from dried blood stains nor from stool sample fungi parasites or the purification of RNA The application of the kit for isolation and purification of viral DNA has not been evaluated Trademarks InviMag RTP Thermo Eppendorf Registered marks trademarks etc used in this document even when not specifically marked as such are not to be considered unprotected by law InviMag and RTP 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 r
37. te During lysis see Preliminary Steps to process the sample onto the KingFisher System page 17 and follow the instructions Lysis at 95 C for 10 min in a thermomixer Place the Extraction Tube in a thermomixer and incubate at 95 C for 10 min continuously shaking increases the lysis efficiency After lysis centrifuge the sample at max speed for 1 min to spin down unlysed material DNA Binding After centrifugation transfer app 450 ul of the supernatant into the Tube A of the KingFisher tube and add 400 ul of Binding Buffer B6 and 20 ul MAP Solution A see also below Note Vortex the tube MAP Solution A vigorously before use Start the program see instructions on page 17 15 InviMag Bacteria DNA Mini Kit KFmL 0515 Protocol 7 Isolation of bacterial DNA from urine samples Please read the instructions carefully and conduct the prepared procedure Attention Please be aware that you have to prepare the Binding Buffer B6 see instruction page 11 Sample Collection Centrifugation of the collected urine sample 15 50 ml for 15 min at 1 300 x g 3500 rpm Decant the supernatant carefully and resuspend the pellet with 3 ml of 1x PBS Centrifuge for 5 min at 1 300 x g 3500 rpm Decant the supernatant carefully It is important to remove the supernatant completely Residual amounts of liquid will have a negative influence on the further extraction procedure Lysis at 65 C for 10 min in a thermomixer Resuspe
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