InviMag Plant DNA Mini Kit KFmL
Contents
1. 535 5053 7 InviMag Plant DNA Mini Kit KF96 0515 Product characteristic of the InviMag Plant DNA Mini Kit KF96 5 25 ug depends on the up to 100 mg plant material kind and amount of starting material about 30 min without lysis The InviMag Plant DNA Mini Kit KF96 is designed for a semi automated preparation of genomic DNA from up to 100 mg of plant material using magnetic beads and the KF96 or KFflex96 workstation The isolation process is based on the patented InviMag technology The DNA isolation process relies on the interaction of nucleic acids with coated magnetic particles at adapted buffer conditions The KF96 KFflex96 instrument performs all purification steps of the DNA purification procedure automatically except the plant sample preparation lysis and initial loading of the system Sample cross contamination and reagent cross over is effectively eliminated by the provided assay file The KingFisher instrument uses magnetic rods to transport the DNA bound to magnetic particles through the various assay phases like binding washing drying and elution The volume of buffers and other liquids required for DNA isolation is reduced to a minimum To achieve optimal lysis conditions and high yields the plant samples are first mechanically disrupted followed by a lysis step in an optimized buffer system at elevated temperature After lysis a binding step is performed in which the DNA is bound to the magnet2. Sealing Foils Prefilter Plate Initial steps Plastic to be supplied by user see order information 2 0 ml Deep Well Plate KF 96 Tip Comb for DW magnets 200 ul Elution Plate 7437300150 7437300250 210 m 2x1 5 ml 10 5 ml 9 ml 36 ml final volume 30 ml final volume 120 ml 2x 1 1 ml 10 5 ml 80 ml 3 x 80 ml final volume 160 ml final volume 3 x 160 ml 60 ml 4x60 ml final volume 200 ml final volume 4 x 200 ml Add 1 5 ml distilled water to each Proteinase K tube mix thoroughly until completely dissolving and store at 20 C Add 80 ml of 96 100 ethanol to the bottle Wash Buffer Add 140 ml of 96 100 ethanol to the bottle Wash Buffer Il mix thoroughly and always keep the bottles firmly closed shaking by inverting for 1 several times Add 21 ml 99 7 Isopropanol to the the Binding Buffer A Mix by Binding Buffer A Mix by intensive intensive shaking by inverting for 1 min Shortly before use mix by inverting inverting several times Dilute Proteinase K in 10 5 ml of ddH O mix thoroughly until completely dissolving and store at 20 C 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 Add 84 ml 99 7 Isopropanol to min Shortly before use mix by Elution and Tip Plate are identically Use one provided Elution Plate as a Tip
3. 200 ul Binding Buffer A 20 ul SNAP Solution to a 2 ml Deep Well Plate Washing Plate 1 Add 800 ul Wash Buffer I to a 2 ml Deep Well Plate Washing Plate 2 Add 800 ul Wash Buffer Il to a 2 ml Deep Well Plate Washing Plate 3 Add 800 ul Wash Buffer Il to a 2 ml Deep Well Plate Elution Plate Add 100 ul Elution Buffer to the 200 ul Elution Plate 3 Choose either the assay file InviMag Plant KF96 KF96 assay file or InviMag Plant KFflex96 KFflex96 assay file on the display of the corresponding KingFisher instrument and press the START button 4 Insert the prefilled plates onto the right positions of the KingFisher instrument by following the specifications shown on the instrument display and confirm every loading step with the START button When all prefilled plates are loaded press the START button to initialize the assay file The assay file will start at the Binding Step From this point the instrument will continue with the purification process without any further user interaction 12 InviMag Plant DNA Mini Kit KF96 0515 The following extraction steps run automatically on the KingFisher System 1 Binding of the DNA Automatically sample mixing for 5 min SNAPs separation Transfer of the SNAPs to Washing Plate 1 2 First Washing Automatically sample mixing for 1 5 min SNAPs separation Transfer of the SNAPs to Washing Plate 2 3 Second Washing Automatically sample mixing for 1 min SNAPs se
4. Plate InviMag Plant DNA Mini Kit KF96 0515 Symbols MAD Manufacturer LOT Lot number Catalogue number gt lt Expiry date li Consult operating instructions p4 Temperature limitation Q Do not reuse Storage All buffers and kit components of the InviMag Plant DNA Mini Kit KF96 except dissolved Proteinase K and SNAP Solution should be stored at room temperature and are stable for at least 12 months at these conditions Proteinase K Dissolved Proteinase K must be stored at 20 C We recommended preparing aliquots in order to avoid multiple freezing and thawing cycles because this can lead to decreased enzymatic activity SNAP Solution The magnetic particles should be stored at 2 8 C Wash Buffer I Wash Buffer Il Binding Buffer A Buffers charged with either ethanol or isopropanol should be stored at room temperature and must be sealed accordingly If any precipitates are visible within the provided solutions solve them by carefully warming up to 30 C Room temperature RT is defined as range from 15 30 C Quality control and product warranty STRATEC Molecular warrants the correct function of the InviMag Plant DNA Mini 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 the manual STRATEC Molecular will check the lot and if STRATEC Molecular inves
5. Contamination of the liquid waste with residual infectious materials is highly unlikely but cannot be excluded completely Therefore the liquid waste has to be considered infectious and be handled and discarded accordingly to local safety regulations European Community risk and safety phrases for the components of the InviMag Plant DNA Mini Kit KF96 to which they apply are listed below as follows Lysis Buffer P Proteinase K gt OD danger danger H319 P305 351 338 H315 319 334 335 P280 305 351 338 310 405 Wash Buffer D warning H302 312 332 412 EUH032 P273 H315 Causes skin irritation H319 Causes serious eye irritation H334 May cause allergy or asthma symptoms or breathing difficulties if inhaled H335 May cause respiratory irritation 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 P280 Wear protective gloves protective clothing eye protection face protection P305 P351 P338 If in eyes Rinse cautiously with water for several minutes Remove contact lenses if present Continue rinsing P310 Immediately call a POISON CENTER or doctor physician P405 Store locked up P273 Avoid release to the environment Emergency medical information can be obtained 24 hours a day from infotrac outside of USA 1 352 323 3500 inside of USA 1 800
6. I smnsssssessressrssrerrrsrrsrrrrrrrrrrrrrr narr rr ran rr r rerna nr nanna nn 10 Scheme of the InviMag Plant DNA Mini Kit KF96 s umusenssrsrrsrrsrrsrrserserserrrrrrrrrr rr ner er renen 200200 an 11 LYSIS PTO CEO UNC S ira raticedtd esheets rea E S 12 Protocol Isolation of genomic DNA from up to 100 mg of plant material cseeeeeee 12 Starting a Run on a KF96 KFflex96 INStrUMENT eee ce ccc ceeeeceeeeceeeeeeeeeeseeeeeaeeesaaeeeseeeesseeeas 12 For self programming of the KF96 KFflex96 INStrUMENT ccc cceecceeceeeeeeeeeeeeeeeeaeeeaeeeaeeseeenes 14 TROUDICS MOOG teisutenousseuadjeusincusuenssen EEE EA Ea aE E ERRAT 16 ADENO a aa E T TT T R Fehler Textmarke nicht definiert General notes onnandino DNA serrara sluss 18 Oraernng ATOM UO airc a E a ieee eee A 19 Ordering information KingFisher 96 and consumables cccseceeeeeeeeeeeeeeeeeeseeeeseeeeeseeesaeeeess 19 InviMag Plant DNA Mini Kit KF96 0515 Kit contents of InviMag Plant DNA Mini Kit KF96 Store the SNAP Solution at 2 8 C Store diluted Proteinase K at 20 C Store all other kit components at room temperature 96 extractions 5 x 96 extractions Catalogue No 7437300100 7437300200 Proteinase K working solution 2 x 1 5 ml 10 5 mi PEST 9 ml 36 ml SNAP Solution 80 ml 3 x 80 ml Wash Buffer final volume 160 ml final volume 3 x 160 ml 60 ml 4x 60 ml Wash Buffer II final volume 200 ml final volume 4 x 200 ml E
7. KingFisher 96 tip comb for a PCR magnet head plates 8 x 10 pcs box KingFisher 96 tip comb for KF magnets plates 10 x 10 pcs box KingFisher 96 tip comb for DWP magnets DWP 10 x 10 pcs box KingFisher 96 KF plate 200 ul 48 plates box Microtiter deep well 96 plate 2 ml 50 plates box 19 InviMag Plant DNA Mini Kit KF96 0515 Stratecee molecular STRATEC Molecular GmbH Robert Rossle Str 10 13125 Berlin Germany Phone 49 30 94 89 29 01 Fax 49 30 94 89 29 09 E mail info berlin stratec com www stratec com 1G3d02 05 2015
8. Propanol Rotipuran gt 99 7 p a ACS ISO Order Nr A3928 Order no 59304 1L F Order no 6752 10 InviMag Plant DNA Mini Kit KF96 0515 Scheme of the InviMag Plant DNA Mini Kit KF96 SS Transfer the crushed plant material into 1 5 ml reaction tubes not provided or a 96 DWP not provided and add 400 ul Lysis Buffer P and 20 ul Proteinase K Incubate the lysis mixture for 30 min at 60 C while continuously shaking After lysis a filtration step of the lysis mixture into the Binding Plate prefilled with 200 ul Binding Buffer A follow preparing instructions and 20 ul SNAP Solution is performed DNA binds to magnetic particles Magnetic separation Washing of the particle fixed genomic DNA with 800 ul Wash Buffer and twice with 800 ul Wash Buffer Il Magnetic separation and drying step Elution of genomic DNA with 100 ul Elution Buffer Magnetic Separation and bead removal Pure DNA 14 InviMag Plant DNA Mini Kit KF96 0515 Lysis Procedures Protocol Isolation of genomic DNA from up to 100 mg of plant material Please read the instructions carefully and conduct the prepared procedure Homogenization of the starting material Homogenize up to 100 mg of plant material by use of a pestle and mortar in combination with liquid nitrogen Commercially available equipment for homogenization bead mill etc can be used too Transfer the homogenized starting material either into 1 5 ml reaction tubes not provid
9. Sstratecee molecular 3 User manual InviMag Plant DNA Mini Kit KF96 for use on KingFisher 96 and KingFisher Flex Thermo Fisher for automated purification of genomic DNA from up to100 mg plant material and food of vegetable origin with magnetic beads 7437300X0O pos STRATEC Molecular GmbH D 13125 Berlin T Instruction for InviMag Plant DNA Mini Kit KF96 The InviMag Plant DNA Mini Kit KF96 is the ideal tool for isolation and purification of DNA from up to 100 mg plant material or food from plant origin with the patented InviMag technology using a KF96 KFflex96 instrument The kit is neither validated for the isolation of genomic DNA from cultured or isolated cells from blood stool samples swabs dried blood stains or cell free body fluids like cerebrospinal fluid synovial fluid and urine nor from bacteria fungi parasites or purification of RNA Trademarks InviMag Invisorb Registered marks trademarks etc used in this document even when not specifically marked as such are not to be considered unprotected by law The Invisorb technology is covered by patents and patent applications US 6 110363 US 6 043 354 US 6 037 465 EP 0880535 WO 9728171 WO 9534569 EP 0765335 DE 19506887 DE 10041825 2 WO 0034463 InviMag and Invisorb are registered trademarks of STRATEC Biomedical AG The PCR process is covered by US Patents 4 683 195 and 4 683 202 and foreign equivalents owned by Hof
10. d 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 InviMag Plant DNA Mini Kit KF96 0515 Safety information When and while working with chemicals always wear a suitable lab coat disposable gloves and protective goggles Avoid skin contact 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 Plant DNA Mini Kit KF96 procedures for residual infectious materials
11. ed or use a 96 DWP not provided and add 400 ul of Lysis Buffer P and 20 ul of Proteinase K to each sample Incubate the mixtures at 60 C for 30 min while continuously shaking During lysis prefill all plates with the required buffers and appropriate volumes see Starting a run page 12 After lysis set up the Prefilter plate onto the Binding Plate 2 0 ml Deep Well Plate and transfer the lysis mixtures into the filter plate Incubate for 2 3 min at room temperature or until all lysates have passed the filter plate The lysed samples have now been transferred into the Binding Plate Remove the filter plate and start the run on the KF instrument see below Important The kit will also co purify RNA beside DNA For the elimination of RNA if required add 20 ul RNase A 10 mg ml to the Lysis Buffer P prior before starting the lysis procedure Starting a Run on a KF96 KFflex96 instrument Attention Please be aware that you have to prepare the Binding Buffer A see instruction page 10 Note Before starting the purification process with the KingFisher instrument please carefully read the manufacturer s manual Resuspend Vortex the magnetic particles SNAP Solution thoroughly before use 1 Switch on the KingFisher instrument 2 Prefill all required plates as described below Tip Plate Place the KF96 Tip Comb for DW magnets on a Tip Plate Use one provided Elution Plate as Tip Plate These are identical Binding Plate
12. ed material is lysed at denaturing non chaotropic conditions at elevated temperatures in presence of Lysis Buffer P and Proteinase K Because unlysed material has to be removed after lysis by filtration the lysis procedure has to be performed externally The filtration step is required because in most cases a centrifugation step will not be able to remove very small debris especially if a mortar and pistil was used for disrupting Binding of the DNA After addition of Binding Buffer A and SNAP Solution to the lysate optimal binding conditions are adjusted and the genomic DNA is bound to the magnetic particles Removing residual contaminants Contaminants are efficiently removed using Wash Buffer and Wash Buffer Il while the DNA remains bound to the magnetic beads Elution The DNA is finally eluted in Elution Buffer The eluted DNA is ready to use in different subsequent downstream applications like o PCR RAPD AFLP analysis o microsatellite analysis o genotyping o enzymatic restriction digestion Yield and quality of genomic DNA The amount of purified DNA derived by the InviMag Plant DNA Mini Kit KF96 procedure from plant materials depends on the sample source transport conditions storage and sample age The overall yield and quality of the isolated genomic DNA is suitable for any detection system The PCR process is covered by US Patents 4 683 195 and 4 683 202 and foreign equivalents owned by Hoffmann La Roc
13. fmann La Roche AG 2015 STRATEC Molecular all rights reserved InviMag Plant DNA Mini Kit KF96 0515 Table of content Kit contents of InviMag Plant DNA Mini Kit KF96 ssnmensersersersrssrssersrssesrerrerser ses rss soner renen a non on 3 Kit contents of InviMag Plant DNA Mini Kit KF96 W o PlaStiC ssserserorsrssrssrsersreersrssrerr ronne 4 DOS renee Run Se nS On teeta aoe Te Rene AeA ee ee RE eR EL Nee eee Ae ete ee Ee 5 Storage enes dr ent dor dura SSR E ean NERE 5 mended USE eanan E SERNER ESKS NIGER ONES NRA NA SST STE 6 FPTOGUCT USE imitato certedencacnientasonndensansranessnciienehakbauteoanibenaantadnatdabeannientadostdeldasmngastnsienabakbaurasansionts 6 SAIOLY NOMO sossar alee ee eer ie re a a rat ha Nr eats 7 Product characteristic of the InviMag Plant DNA Mini Kit KF96 0 0 cccecseseceseeesesesesereeeseeeeees 8 Sampling and storage of Starting MateriQl sssnnsmssssssssessrrersrresrrerrrnrrrn ere rr ers nns r rr rr rr RR RR RAR RR R RKS RR Kn rann 9 PANCE aNd IP rOCS CUP G sereia vilse instansen a a a venta a a 9 Yield and quality of genomie DNA 4 ssssimuenisesnvsndsiusrisbesde a tered ne Rare 9 Important points before starting a PrOtOCOl s sessersrrsrrrrrrrrrsrrorrrrrnrrrrrr ren ren rr ren rar rr rr rr rr RR Rn rn nen 10 Preparing reagents and buffers smesssssessessrrrrresrarrrsrrrrrrrrrrr rr rens narr arr rr rr RR ARR KSR KKR KSR KKR KSR KKR KR RKS R nn 10 Reagents and equipment to be supplied DY USC
14. he AG 9 InviMag Plant DNA Mini Kit KF96 0515 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 Change pipet tips between liquid transfers To avoid cross contamination we recommend the use of aerosol barrier pipet 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 This kit should only be used by trained personnel O O O Preparing reagents and buffers Before starting a run bring all reagents to room temperature Gently mix and redissolve any precipitates by warming up to 30 C Swirl gently to avoid foaming Lysis Buffer P and Elution Buffer are ready to use Add the required amount of ddH20 to the reaction tube containing the Proteinase K Vortex f
15. ic particles followed by several washing steps before the pure DNA is finally eluted The purified and high quality DNA can be stored at 20 C for subsequent use and is ready to use for subsequent downstream applications like o PCR o Genotyping o Restriction digestion No toxic or hazardous chemicals are used For the isolation of DNA from single plant samples STRATEC Molecular offers the Invisorb Spin Plant Mini Kit as well as 96 well kits for use in a centrifuge For further information please contact 49 0 30 9489 2901 or 2910 in Germany and 49 0 30 9489 2907 from foreign countries or ask your local distributor g InviMag Plant DNA Mini Kit KF96 0515 Sampling and storage of starting material Harvested plant samples can be stored at room temperature for up to 2 3 hours For short term storage up to one week samples may be stored at 2 8 C For long term storage we recommend freezing samples at 20 C or 80 C Multiple thawing and freezing cycles before isolating the DNA should be avoided because this can lead to degraded DNA and reduced yields 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 Lysis Samples are disrupted by using a mixer mill bead mill or by mortar and pistil in combination with liquid nitrogen Afterwards the disrupt
16. ing 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 C Preheat Postmix Collect count Collect time s Washing Plate 3 Release time speed Tip Plate No 00 00 15 Fast 00 05 00 Medium No No 3 10 No 00 00 10 Fast 00 01 30 Fast No No 3 5 No 00 00 10 Fast 00 01 00 Fast No No 3 5 No 00 00 10 Fast 00 01 00 Fast No No 3 5 00 05 00 Outside well tube No 00 00 10 Medium 00 10 00 Slow 00 00 30 Fast 15 InviMag Plant DNA Mini Kit KF96 0515 Troubleshooting Probable cause Comments and suggestions low amount of extracted DNA low concentration of extracted DNA degraded sheared DNA DNA does not perform well in downstream applications e g real time PCR or PCR low Azgo Azgo ratio from UV measurement eluted DNA is brown colored insufficient lysis incomplete elution low amount of SNAP Solution too much Elution Buffer incorrect storage of starting material incorrect storage of starting material old material ethanol carryover during elution salt carryover during elution small part of the magnetic particles are left in the elution 16 inc
17. ith caution Avoid vigorous pipetting Pipetting of 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 from the plant material depends on sample source transport conditions storage and age of the sample 18 InviMag Plant DNA Mini Kit KF96 0515 Ordering information InviMag Plant DNA Mini Kit KF96 7437300100 1 x 96 preps InviMag Plant DNA Mini Kit KF96 7437300200 5 X 96 preps InviMag Plant DNA Mini Kit KFmL 2437110100 15 purifications InviMag Plant DNA Mini Kit KFmL 2437110200 75 purifications Invisorb DNA Plant HTS 96 Kit C 7037300200 2 x 96 preps Invisorb DNA Plant HTS 96 Kit C 7037300300 4 x 96 preps Invisorb DNA Plant HTS 96 Kit C 7037300400 24 x 96 preps Invisorb Spin Plant Mini Kit 1037100200 50 purifications Invisorb Spin Plant Mini Kit 1037100300 250 purifications Possible suppliers for lsopropanol Sten ot ae 3 2 Propanol f r die Molekularbiologie 2 Propanol BOUPUED I e pAn ASSA Order no A3928 Order no 59304 1 L F Order no 6752 Ordering information KingFisher 96 and consumables Cat no 5400500 24073430 97002514 97002524 97002534 97002540 95040450 Description KingFisher 96 magnetic particle processor 100 240V 50 60Hz including one magnetic head KingFisher 96 head for Deep Well Plates
18. lution Buffer 1 piece 5 pieces magnets Initial steps Add 1 5 ml distilled water to each Dilute Proteinase K in 10 5 ml of Proteinase K tube mix ddH O mix thoroughly until thoroughly until completely completely dissolving and store dissolving and store at 20 C at 20 C Add 80 ml of 96 100 ethanol to Add 80 ml of 96 100 ethanol to the bottle Wash Buffer each bottle Wash Buffer Add 140 ml of 96 100 ethanol Add 140 ml of 96 100 ethanol to to the bottle Wash Buffer Il mix each bottle Wash Buffer II mix thoroughly and always keep the thoroughly and always keep the bottles firmly closed bottles firmly closed Add 21 ml 99 7 Isopropanol to Add 84 ml 99 7 Isopropanol to the Binding Buffer A Mix by the Binding Buffer A Mix by intensive shaking by inverting for intensive shaking by inverting for 1 min Shortly before use mix by 1 min Shortly before use mix by inverting several times inverting several times Elution and Tip Plate are identically Use one provided Elution Plate as a Tip Plate InviMag Plant DNA Mini Kit KF96 0515 Kit contents of InviMag Plant DNA Mini Kit KF96 w o plastic Store the SNAP Solution at 2 8 C Store diluted Proteinase K at 20 C store all other kit components at room temperature 96 extractions 5 x 96 extractions Catalogue No Lysis Buffer P Proteinase K working solution Binding Buffer A SNAP Solution Wash Buffer Wash Buffer Il Elution Buffer
19. nd 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 develooment 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 hg SPE MS Windows XP Pro with SP3 Windows Vista SP2 Windows 7 Disk space 500 MB free disk space Processor Intel Pentium gt 1 GHz Memory 1 GB RAM Serial ports available 1 for KFML connection USB ports available 1 for KF96 KFflex96 KFDuo connection Pointing device Mouse or equivalent is required CD ROM drive XVGA monitor with at least 1024x768 resolution and at least a 16 bit color Monitor color settings i onitor color setting environment If the actual Windows Service Packs a
20. olume ul F 420 00 0 dT pot Well volume ul 800 Well volume ul 800 Well volume ul 800 Well volume ul 100 King Fisher 96 KF plate Total reagent volume pl Microtiter DW 96 plate Total reagent volume pl Microtiter DW 96 plate Total reagent volume ul Microtiter DW 96 plate Total reagent volume ul Microtiter DW 96 plate Total reagent volume ul King Fisher 96 KF plate Total reagent volume ul Type Type Sample Reagent Reagent Type Reagent Type Reagent Type Reagent Type Reagent The protocol does not contain dispensed reagents 14 InviMag Plant DNA Mini Kit KF96 0515 Steps data Tip 1 ig 5 Pick Up Binding Step Beginning of step Mixing heating End of step Washing Step I Beginning of step Mixing heating End of step Washing Step 2 Beginning of step Mixing heating End of step Washing Step 3 Beginning of step Mixing heating End of step Drying Elution Beginning of step Mixing heating End of step Bead Removal Leave 96 DW tip comb Tip Plate Binding 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 Heat
21. or 5 s and keep the solution on ice Store unused and diluted Proteinase K at 20 C 1x 96 DNA extractions Add 21 ml 99 7 lsopropanol to the Binding Buffer A Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times Dilute Proteinase K by addition of 1 5 ml of ddH20 mix thoroughly until completely dissolving and store at 20 C Add 80 ml of 96 100 ethanol to the bottle Wash Buffer Add 140 ml of 96 100 ethanol to the bottle Wash Buffer II Mix thoroughly and always keep the bottle firmly closed 5x 96 DNA extractions 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 Dilute Proteinase K by addition of 10 5 ml of ddH2O mix thoroughly until completely dissolving and store at 20 C Add 80 ml of 96 100 ethanol to the bottle Wash Buffer I Add 140ml of 96 100 ethanol to the bottle Wash Buffer Il Mix thoroughly and always keep the bottle firmly closed Reagents and equipment to be supplied by user o Measuring cylinder 250 ml o Vortexer o Pipette and pipette tips o 96 100 ethanol o Disposable gloves o Isopropanol O ddH O The InviMag Plant DNA Mini 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 Applichem Sigma 2 Propanol 3 2 Propanol f r die Molekularbiologie 2
22. paration Transfer of the SNAPs to Washing Plate 3 4 Third Washing Automatically sample mixing for 1 min SNAPs separation 5 Drying Drying of SNAPs outside Washing Plate 3 for 5 minutes Transfer of the SNAPs to the Elution Plate 6 Elution of the DNA Incubation of SNAPs for 10 minutes at 65 C while continuously mixing SNAPs separation Removal of SNAPs into Washing Plate 3 disposal Important Notes 1 After finishing the extraction protocol the Elution Plate contains the extracted DNA Store the DNA at adequate conditions For long term storage we recommend to store the DNA at 20 T 2 If the extracted DNA contains carryover from magnetic particles transfer the DNA into a new 1 5 ml reaction tube and centrifuge at maximum speed for 1 minute Transfer the clear supernatant containing the DNA into a new tube The eluted DNA is ready to use in different downstream applications The eluted DNA can be stored for several weeks at 4 8 C or stored at 20 C for long term storage 13 InviMag Plant DNA Mini Kit KF96 0515 For self programming of the KF96 KFflex96 instrument Reagent info Tip Plate Name Binding Plate Name Crushed sample material in Lysis Buffer P Binding Buffer A SNAP Solution Washing Plate 1 Name Wash Buffer I Washing Plate 2 Name Wash Buffer II Washing Plate 3 Name Wash Buffer II Elution Plate Name Elution Buffer Dispensed reagents Well volume ul Well v
23. pic buffer components For reproducible and high yields appropriate sample storage is essential 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 RNA 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 your results adequate controls for downstream applications should be used Product use limitation The kit is neither suitable for the isolation of DNA from blood serum or plasma bacteria fungi or viruses nor for isolation and purification of 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 conditione
24. re not installed on the corresponding lab computer they can be downloaded from the Microsoft web pages http www microsoft com 17 InviMag Plant DNA Mini Kit KF96 0515 General notes on handling DNA Nature of DNA The length and delicate physical nature of DNA requires careful handling to avoid damage due to shearing and enzymatic degradation Other conditions that affect the integrity and Stability of DNA include acidic and alkaline environments high temperature and UV irradiation Careful isolation and handling of high molecular weight DNA is therefore required to ensure compatibility with various downstream applications Damaged DNA may perform poorly in applications such as genomic Southern Blotting or 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 DNA should be stored at 20 C but storing at 20 C may cause shearing particularly if the DNA is exposed to repeated freezing and thawing cycles Note that the solution in which the nucleic acid is eluted in will affect it s 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 overdrying of genomic DNA after ethanol precipitation We highly recommend to air dry than to use a vacuum although vacuum drying can be used w
25. rease lyses time but prevent too long lyses time because this also decreases the yield Reduce amount of starting material increase volume of Elution Buffer increase time of elution step mix SNAP Solution thoroughly before addition elute the DNA with a lower volume of Elution Buffer don t use less than 100 ul ensure that storage of starting material is correct avoid repeated freezing and thawing cycles of the material ensure that the storage of starting material was correct avoid multiple freezing and thawing cycles of the sample material ensure that the starting material is fresh or stored at appropriate conditions long term storage at 20 C old material often contains degraded DNA increase drying time for evaporation of ethanol check the Wash Buffers for salt precipitates If any precipitates are visible solve them by carefully warming up to 30 C ensure that the Wash Buffers are equilibrated at room temperature centrifuge at full soeed for 1 min and transfer supernatant to a new tube InviMag Plant DNA Mini Kit KF96 0515 Appendix KingFisher Bindlt Software 3 2 or higher versions Bindlt software 3 2 or higher versions were and may be used to create assay files for the KFmL KF96 KFflex96 or KF Duo instruments The provided assay file s can either be transferred onto the corresponding workstation s or be started directly from within the Bindlt software after assay import Please keep in mi
26. tigates 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 Plant DNA Mini 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 of InviMag Plant DNA Mini 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 InviMag Plant DNA Mini Kit KF96 0515 Intended use The InviMag Plant DNA Mini Kit KF96 is designed for a fully automated preparation of genomic DNA from up to 100 mg plant or food plant origin material using the patented InviMag technology in combination with a KF96 KFflex96 instrument The whole process is based on a patented bead technology used for isolation of genomic DNA by binding the nucleic acid onto magnetic particles in absence of chaotro
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