InviMag Universal RNA Mini Kit/ KF96
Contents
1. STEPS BIND Step parameters 18 e Name Binding e Plate Binding Plate Beginning of step e Premix Yes Bind parameters e Bind time 5min Os speed Slow End of step e Collect beads Yes count 5 WASH 1 Step parameters e Name Washing Step 1 e Plate Wash 1 Beginning of step e Release Yes time 30s speed Medium Wash parameters e Wash time 2min Os speed Medium End of step e Collect beads Yes count 3 WASH2 Step parameters e Name Washing Step 2 e Plate Wash 2 Beginning of step e Release Yes time 30s speed Medium Wash parameters e Wash time 1min Os speed Medium End of step e Collect beads Yes count 3 WASH 3 Step parameters e Name Washing Step 3 e Plate Wash 3 Beginning of step e Release Yes time 30s speed Medium Wash parameters e Wash time 1min Os speed Medium End of step e Collect beads Yes count 3 DRY Step parameters e Name Drying e Plate Wash 3 e Dry time 7min Os InviMag Universal RNA Mini Kit KF96 0515 e Tip position Outside well ELUTION Step parameters e Name Elution e Plate Elution Plate Beginning of step e Release Yes time 30s speed Medium Elution parameters e Elution time 10min Os speed Slow e Heating No Remove beads e Remove beads Yes collect count 5 disposal plate Wash 3 19 InviMag Universal RNA Mini Kit2. 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 1A5001 05 2015
3. 1 5 ml reaction tube centrifuge at maximum speed for 1 min and transfer the RNA containing supernatant into a new tube 17 InviMag Universal RNA Mini Kit KF96 0515 For self programming of the KingFisher 96 instrument Protocol InviMag Uni RNA KF96 PROTOCOL PROPERTIES Name InviMag Uni RNA KF96 Protocol template version 2 6 0 Instrument type KingFisher 96 Description KingFisher 96 Thermo Electron protocol for Isolation of total RNA from cells or tissue with the InviMag Universal RNA Mini kit Kit InviIMAG Universal RNA Mini Kit KF96 KFflex96 Plate layouts Tip Plate Binding Plate Wash 1 Wash 2 Wash 3 Elution Plate PLATE LAYOUTS TIP PLATE Plate type KingFisher 96 plate Plate change message Change Tip Plate A EMPTY BINDING PLATE Plate type Thermo DW Plate change message Change Binding A volume 505 name Lysed Sample volume 400 name Ethanol volume 20 name SNAP Solution WASH 1 Plate type Thermo DW Plate change message Change Wash 1 A volume 800 name Wash Buffer R1 WASH 2 Plate type Thermo DW Plate change message Change Wash 2 A volume 800 name Wash Buffer R2 WASH 3 Plate type Thermo DW Plate change message Change Wash 3 A volume 800 name Wash Buffer R2 ELUTION PLATE Plate type KingFisher 96 plate Plate change message Change Elution A volume 100 name Elution Buffer R
4. InviMag Universal RNA Mini Kit KF96 0515 Symbols Lot number Catalogue number Expiry date Consult operating instructions Temperature limitation Ox HKA E Do not reuse Storage All buffers and kit contents of the InviMag Universal RNA Mini Kit KF96 except SNAP Solution should be stored 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 SNAP Solution The magnetic beads should be stored at 4 8 C Wash Buffer 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 Quality control and product warranty STRATEC Molecular warrants the correct function of the InviMag Universal RNA 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 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 134
5. KF96 0515 For self programming of the KingFisher Flex 96 instrument Protocol InviMag_Uni_RNA_KFflex96 PROTOCOL PROPERTIES Name InviMag Uni RNA KFflex 96 Protocol template version 3 1 Instrument type KingFisher Flex 96 Description KingFisher Flex 96 Thermo Electron protocol for Isolation of total RNA from cells or tissue with the InviMag Universal RNA Mini kit Kit InviMAG Universal RNA Mini Kit KF96 KFflex 96 Plate layouts Tip Plate Binding Plate Wash 1 Wash 2 Wash 3 Elution Plate PLATE LAYOUTS TIP PLATE Plate type KingFisher 96 plate Reagents lt empty gt BINDING PLATE Plate type KingFisher 96 DW plate Reagents Name Sample Volume ul 505 Type Sample Name EtOH abs Volume ul 400 Type Reagent Name SNAP Solution Volume pl 20 Type Reagent WASHING PLATE 1 Plate type KingFisher 96 DW plate Reagents Name Wash Buffer R1 Volume ul 800 Type Reagent WASHING PLATE 2 Plate type KingFisher 96 DW plate Reagents Name Wash Buffer R2 Volume yl 800 Type Reagent WASHING PLATE 3 Plate type KingFisher 96 DW plate Reagents Name Wash Buffer R2 Volume yl 800 Type Reagent ELUTION PLATE Plate type KingFisher 96 plate Reagents Name Elution Buffer R Volume ul 100 Type Reagent STEPS TIP PLATE Pick Up plate Tip Plate Leave plate Tip Plate BINDING PLATE Beginning of step Precollec
6. Kit KF96 0515 Scheme of the InviMag Universal RNA Mini Kit KF96 Please read protocols carefully prior to the start of the preparation Prefill all KingFisher plates with the needed buffers and appropriate volumes as shown below Tip Plate Place a KF96 Tip Comb for DW magnets on a 200 ul Elution Plate Binding Plate Add 400 ul Ethanol and 20 ul SNAP Solution Washing Plate 1 Add 800 ul Wash Buffer R1 to 2 0 ml Deep Well Plate Washing Plate 2 Add 800 ul Wash Buffer R2 to 2 0 ml Deep Well Plate Washing Plate 3 Add 800 ul Wash Buffer R2 to 2 0 ml Deep Well Plate Elution Plate Add 100 ul Elution Buffer R to a KF96 Plate same size as Tip Plate Sample Lysis o Transfer the sample into a 1 5 ml reaction tube and add 500 ul LysisBuffer TR and 5 ul DTT 1 M stock solution not provided Disrupt the sample by use of a mortar and pestle or a comparable electronic device o After lysis centrifuge each sample for 1 min at 13000 rpm table centrifuge and transfer the supernatant to the Binding plate containing 400 ul ethanol and 20 ul SNAP solution o Start the run bei either choosing the program InviMag RNA Universal KF96 or InviMag RNA Universal KFflex 96 and follow the instructions shown on the KingFisher instrument display RNA Binding to magnetic beads while DNA is excluded Magnetic separation Washing of the particle fixed RNA Magnetic separation Elution of RNA Magnetic Separation Pure
7. RNA art E Elution Plates KF96 and Tip Plates are identically Use one provided Elution Plate as a Tip Plate 12 InviMag Universal RNA Mini Kit KF96 0515 Protocol 1 Extraction of total RNA from fresh cultured cells or frozen cell pellets Please read the instructions carefully and conduct the prepared procedure Total RNA extraction from human and animal cell culture 1 Harvesting cells Cells grown in Spin down up to 1x10 cells for 5 min at 1 500 rpm Discard the suspension supernatant and remove all media completely In large culture vessels dishes gt 35 mm flasks gt 12 5 cm detach cells by trypsination Transfer the cells into a centrifugation tube and sediment by centrifugation at 1500 rom Cells grown ina for 5 min Remove the supernatant completely monolayer In small culture vessels 96 24 12 6 well plates 35 mm dishes 12 5 cm flasks discard the media completely and continue with the lysis immediately Important Incomplete removal of the cell culture media will inhibit the lysis and dilution of the lysate will affect the binding of RNA to the magnetic beads 2 Cell Disruption a Cell pellet To loosen the cell pellet flick the tube and add Lysis Solution TR with DTT see tab 1 DTT is not supplied within the kit Mix thoroughly by pipetting up and down until no cell clumps are visible Incubate for 2 min at room temperature before proceeding with the next step Ce
8. Universal RNA Mini Kit KF96 in different package sizes The procedure of the InviMag Universal RNA Mini Kit KF96 is optimized for the isolation of RNA from up to 40 mg of fresh or frozen tissue sample from up to 5 x 10 cells and from leukocyte pellets derived from up to 1 5 ml blood 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 diagnostic results adequate controls for downstream applications should be used Product use limitation The kit is neither validated for the isolation of RNA from blood stains stool samples bacteria fungi plants or viruses nor for purification of DNA 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 p
9. Values up to 2 3 are routinely obtained for pure RNA in 10 mM Tris HCl pH 7 5 with some photospectrometers 25 InviMag Universal RNA Mini Kit KF96 0515 Ordering information Product Package size Catalogue No InviMag Universal RNA Mini Kit KF96 1 x 96 preparations 7460300100 InviMag Universal RNA Mini Kit KF96 5 x 96 preparations 7460300200 InviMag Universal RNA Mini Kit KF96 w o plastic 1 x 96 preparations 7460300150 InviMag Universal RNA Mini Kit KF96 w o plastic 5 x 96 preparations 7460300250 KingFisher 96 and consumables KingFisher 96 Magnetic Particle Processor 100 240V 50 60Hz 5400500 KingFisher 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 Related products Package size Catalogue No InviTrap Spin Universal RNA Mini Kit 50 preps 1060100200 InviTrap Spin Universal RNA Mini Kit 250 preps 1060100300 InviTrap Spin Cell RNA Mini Kit 50 preparations 1061100200 InviTrap Spin Cell RNA Mini Kit 250 preparations 1061100300 InviTrap Spin Tissue RNA Mini Kit 50 preparations 1062100200 InviTrap Spin Tissue RNA Mini Kit 250 preparations 1062100300 26 InviMag Universal RNA Mini Kit KF96 0515 Sstratecee molecular
10. after disruption Contaminating genomic DNA and other cellular components of high molecular weight are sheared to form a homogenous lysate Incomplete homogenization results in an inefficient binding of RNA to the magnetic beads and therefore significantly reduces yields It is possible to use a commercially available bead mill in combination with or without beads for the disruption and homogenization of the starting material Alternatively the starting material can be grinded to a fine powder in liquid nitrogen using a mortar and pestle Some disruption methods simultaneously homogenize the sample while others require an additional homogenization step Rotor stator homogenizers and bead mills are used for simultaneously disruption of samples In contrast the use of a mortar and pestle only drisrupt the sample To achieve a complete homogenization a separate step is needed This can be accomplished by use of a shredder spin column or a 20 gauge needle a Disruption and homogenization using rotor stator homogenizer Using a rotor stator homogenizer in combination with Lysis Solution TR and DTT see Table 1 page 13 disrupts and simultaneously homogenizes in 5 90 s depending on the toughness of the sample b Disruption and homogenization using a bead mill e g Gyrator With bead mills tissue samples can be disrupted by rapid agitation in the presence of Lysis Solution TR and DTT see Table 1 page 13 Disruption and simultaneous homog
11. purification of cell RNA using magnetic beads and the KingFisher 96 or KingFisher Flex 96 The DNA is removed during the process Starting Material Yield Time Ratio from 10 40 mg fresh or frozen up to 80 ug depending on the about 60 min A260 A280 tissue used tissue 1 7 2 1 from up to 5 x 10 fresh or frozen up to 20 ug depending on the cells used cells from up to 1 5 ml whole blood up to 6 ug depending on the used blood The RNA isolation process is based on the interaction of nucleic acids with magnetic particles under adapted buffer conditions The KingFisher instrument performs all steps of the RNA purification procedure automatically with only minor user intervention thus allowing safe handling of potentially infectious samples Sample cross contamination and reagent cross overs are effectively eliminated by this automated purification process The KingFisher instrument uses magnetic rods to transfer the RNA binding magnetic particles through the various purification phases DNA removal RNA binding washing and elution The volume of buffers and other liquids necessary for RNA isolation is reduced to a minimum Eliminating the direct liquid handling and increasing the automation level result in a fast reliable and robust technique The overall efficiency speeds up the procedure After cell lysis outside the workstation in the optimized Lysis Solution TR and removal of the DNA optimal binding conditions are adjusted by additio
12. 5 ml of cooled Buffer EL to the cell pellet vortex shortly and centrifuge at 1 700 x g 3 000 rpm for 5 min at 4 C Carefully remove the supernatant completely Note Incomplete removal of the supernatant will interfere with lysis and subsequent binding of the RNA to magnetic particles resulting in lower yield and purity 2 Leukocyte disruption Important Before starting with step 2 vortex the Lysis Solution TR vigorously and avoid contaminations with RNase Resuspend the cell pellet from step 1 in 500 ul Lysis Solution TR with DTT see Table 2 Vortex or pipet up and down several times to solve the cell pellet completely Incubate for 2 min before proceeding with the next step Centrifuge the suspension for 1 min at maximum speed e g 13 000 rpm on a table centrifuge Carefully transfer the supernatant to the prepared Binding Plate without disturbing the pellet Gyrator UNIPREP universal 3 D Vortexer for Microtubes from UniEquip GmbH phone 49 0 89 8575200 phone 49 0 89 8575205 Fax 49 0 89 8561304 email info uniequip de Please refer to suppliers guideline for further details 15 InviMag Universal RNA Mini Kit KF96 0515 Starting a run on a KF96 or KFflex 96 instrument I Preliminary Steps to process the sample onto the KingFisher System Important 1 For working with the KF96 or KFflex 96 system please carefully read the documents supplied by the instrument manufacturer Turn on the KF96 KF
13. 85 certified Quality Management System the performance of all components of the InviMag Universal RNA 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 Universal RNA 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 2903 or contact your local distributor InviMag Universal RNA Mini Kit KF96 0515 Intended use The InviMag Universal RNA Mini Kit KF96 is designed for semi automated extraction and purification of RNA DNA is excluded from up to 96 samples using magnetic beads and the KingFisher 96 or KingFisher Flex 96 instrument The supplied nucleic acid isolation protocols are capable for routinely walk away automated preparation of RNA from fresh or frozen cultured cells tissue samples and leukocyte pellets derived from blood For reproducible and high yields an appropriate sample storage is essential See Sampling and storage of the starting material page 8 All utilities reagents and plastic ware necessary for preparation of RNA are provided by the InviMag
14. A sample 180 ul of 10 mM Tris HCl pH 7 0 1 10 dilution o Measure absorbance of diluted sample in a 0 2 ml cuvette RNase free Asgo 0 2 Concentration of the RNA sample 40 ug ml Azgo dilution factor 40 ug ml 0 2 10 80 ug ml Total amount concentration volume of sample in ml 80 ug ml 0 1 ml 8 ug of RNA Purity of RNA The ratio of the readings at 260 nm and 280 nm Azgo Azg0 provides an estimate of the purity of RNA with respect to the contaminants that absorb in the UV such as protein However the Azgo Azgo ratio is influenced considerably by pH Since water is not buffered the pH and the resulting Azgo Azgo ratio can vary greatly Lower pH results in lower Axzgo Azgo ratio and reduced sensitivity to protein contaminations For accurate values it is recommend to measure absorbance in 10 mM Tris Cl pH 7 5 Pure RNA has an Adsgo Azgo ratio of 1 9 2 1 in 10 mM Tris HCl pH 7 5 Always be sure to calibrate the spectrophotometer with the same solution For determination of the RNA concentration however it is recommend diluting the sample in a buffer with neutral pH since the relationship between absorbance and concentration Az o reading of 1 40 ug ml of RNA is based on an extinction coefficient calculated for RNA at neutral pH Wilfinger W W Mackey M and Chomczynski P 1997 Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity Bio Techniques 22 474
15. Sstratecee molecular User manual InviMag Universal RNA Mini Kit KF96 for use on KingFisher 96 and KingFisher Flex Thermo Fisher Scientific for automated purification of total RNA with a magnetic bead based DNA removal from up to 5 x10 human and animal cells up to 1 5 ml whole blood or up to 40 mg tissue sample with magnetic beads 7460300X00 gaai STRATEC Molecular GmbH D 13125 Berlin re Instruction for InviMag Universal RNA Mini Kit KF96 The InviMag Universal RNA Mini Kit KF96 combines the advantages of the innovative Invisorb technology with easy handling of magnetic particles for a very efficient and reliable isolation of nucleic acids with a high purity The RNA binding magnetic particles are characterized by a high surface area uniform size distribution good suspension stability and therefore are highly suitable for high throughput processing The InviMag Universal RNA Mini Kit KF96 is applicable for isolation and purification of RNA from up to 5 x 10 cell pellets fresh or frozen from 10 40 mg tissue or from leucocyte pellets derived from up to 1 5 ml blood whereas the DNA is efficiently removed by the process The kit is designed for an optimal use on the KingFisher 96 or KingFisher Flex 96 workstation from Thermo Scientific The interplay of the RNA extraction and purification chemistry provided by the InviMag Universal RNA Mini Kit KF96 was intensely tested and validated Du
16. a RS 232 full duplex interface Supported operating systems Microsoft Windows 2000 Microsoft Windows XP Professional Disk space 500 MB free disk space Processor Intel Pentium 700 Mhz recommended Memory 220 MB RAM recommended Serial ports available Pointing device Mouse or equivalent is necessary CD ROM drive 1 Monitor color settings SVGA monitor with at least 1024 x 768 resolution and at least a 16 bit color environment Service packs installed Microsoft Windows 2000 Service Pack 4 or greater 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 23 InviMag Universal RNA Mini Kit KF96 0515 General notes on handling RNA RNA is far less stable than DNA It is very sensitive to degradation by endogenous RNases in the biological material and exogenous RNases which are permanently present everywhere in the lab To achieve satisfactory qualitative and quantitative results in RNA preparations contaminations with exogenous RNases has to be reduced as much as possible Avoid handling bacterial cultures cell cultures or other biological sources of RNases in the same lab where the RNA purification is to be carried out All glassware should be treated before
17. anol to each bottle Wash Buffer R1 Add 200 ml of 96 100 ethanol to each bottle Wash Buffer R2 Mix thoroughly and always keep the bottles firmly closed InviMag Universal RNA Mini Kit KF96 0515 Kit contents of InviMag Universal RNA Mini Kit KF96 w o plastic Store the SNAP Solution at 4 C Store all other kit components at room temperature 1 x 96 extractions 5 x 96 extractions Catalogue Number 7460300150 7460300250 Buffer EL conc 2x30 ml 8 x 30 ml Lysis Solution TR 60 ml 260 ml SNAP Solution 2x 1 1 ml 10 5 ml 80 ml 2x 125 ml Wash Buffer R1 final volume 160 ml final volume 2 x 250 ml Wash Buffer R2 40 ml final volume 200 ml 4x 50 ml final volume 4 x 250 ml Elution Buffer R 15 ml 60 ml 1 5 ml Receiver Tubes 2x50 10 x 50 Manual 1 1 Initial steps Add 30 ml Buffer EL to 970 ml pure Add 30 ml Buffer EL to 970 ml pure water 2x Add 80 ml of 96 100 ethanol to the bottle Wash Buffer R1 Add 160 ml of 96 100 ethanol to the bottle Wash Buffer R2 Mix thoroughly and always keep the bottles firmly closed water 8x Add 125 ml of 96 100 ethanol to each bottle Wash Buffer R1 Add 200 ml of 96 100 ethanol to each bottle Wash Buffer R2 Mix thoroughly and always keep the bottles firmly closed Plastic to be supplied by user see order information 2 0 ml Deep Well 4 20 KF96 Tip Comb for 1 5 KingFisher 96 KF 2 10 plate
18. e to the high purity the isolated RNA is ready to use for a broad panel of downstream applications or can be stored alternatively at 80 C for subsequent use The kit is neither suitable for isolation of RNA from blood stains stool samples bacteria fungi plants or viruses nor for purification of DNA Trademarks InviMag Invisorb Registered mark trademarks etc used in this document even when not specifically marked as such are not to be considered unprotected by law The Invisorb technology is covered by patents and patent applications US 6 110363 US 6 043 354 US 6 037 465 EP 0880535 WO 9728171 WO 9534569 EP 0765335 DE 19506887 DE 10041825 2 WO 0034463 InviMag and Invisorb are registered trademarks of STRATEC Biomedical AG The PCR process is covered by US Patents 4 683 195 and 4 683 202 and foreign equivalents owned by Hoffmann La Roche AG 2015 STRATEC Molecular all rights reserved InviMag Universal RNA Mini Kit KF96 0515 Content Kit contents of InviMag Universal RNA Mini Kit KF96 3 Kit contents of InviMag Universal RNA Mini Kit KF96 w o plastic 4 Symbols 5 Storage 5 Quality control 5 Intended use 6 Product use limitation 6 Safety information 7 Product characteristics of the InviMag Universal RNA Mini Kit KF96 8 Sampling and storage of starting material 9 Preparing reagents and buffers 10 Product validation 11 Scheme 12 Protocol 1 Extraction of RNA from fresh cultured cells or
19. enization occurs by shearing and crushing with the beads as they collide with the cells The disruption efficiency is influenced by Size and Composition of beads Ratio of buffer to beads Amount of starting material Speed of the mill Disintegration time O 2 Q O O A mixture of Zirconia beads with a diameter of 7 x 0 7 mm Zirconia Beads I and 2 x 2 4 mm Zirconia Beads Il are the optimal mixture for tissue disruption All other disruption parameters should be determined empirically for each application c Disruption and homogenization using a mortar and pestle To disrupt tissue using a mortar and pestle freeze the sample immediately in liquid nitrogen and grind it to a fine powder Transfer the suspension tissue powder and liquid nitrogen into an RNase free tube Allow the liquid nitrogen to evaporate but do not allow the sample to thaw After evaporation of the liquid nitrogen add 500 ul Lysis Solution TR with DTT see Table 1 page 13 After addition of Lysis solution end of step a b or c vortex or pipet up and down several times to solve the cell pellet completely Incubate for 2 min at RT before proceeding with the next step Centrifuge the suspension for 1 min at maximum speed e g 13 000 rpm on a table centrifuge Carefully transfer the supernatant to the prepared Binding Plate without disturbing the pellet 14 InviMag Universal RNA Mini Kit KF96 0515 Protocol 3 RNA extraction from up to 1 5 ml whole bl
20. flex 96 system 2 Prefill the Deep Well Plates with the appropiate buffers and volumes Notes O Oo Oo Important Notes In case of not starting a run immediately please avoid evaporation of the prefilled buffer components by sealing the Deep Well Plates with a sealing foil or parafilm Mix the bottle with the SNAP Solution by vigorously vortexing before use Use one of the provided Elution Plate as a Tip Comb Plate These plates are identical Tip Plate Binding Plate Washing plate_1 Washing plate_2 Washing plate_3 Elution Plate Place a KF96 Tip Comb for DW magnets on a 200 ul Elution Plate Transfer the supernatant of the lysed sample into the cavity of the Binding Plate prefilled with 400 ul Ethanol and 20 ul SNAP Solution Pipet 800 ul Wash Buffer R1 into the cavities of a Deep Well Plate Pipet 800 ul Wash Buffer R2 into the cavities of a Deep Well Plate Pipet 800 ul Wash Buffer R2 into the cavities of a Deep Well Plate Pipet 100 ul Elution Buffer R into the cavities of the Elution Plate Choose the program InviMag Uni RNA_KF96 KF96 protocol or InviMag Uni RNA_KFflex96 KFflex 96 protocol and press the START button Insert the prefilled plates into the right position of the instrument surface by following the specification given on the display and confirm every step by pressing the START button After all prefilled plates have been loaded press the START button agai
21. frozen cell pellets 13 Protocol 2 Extraction of RNA from fresh or frozen tissues 14 Protocol 3 RNA extraction from up to 1 5 ml whole blood 15 Starting a run on a KingFisher instrument 16 For self programming of the KingFisher 96 system 18 For self programming of the KingFisher Flex 96 system 20 Troubleshooting 22 Appendix 23 General notes on handling RNA 24 Ordering information 26 InviMag Universal RNA Mini Kit KF96 0515 Kit contents of InviMag Universal RNA Mini Kit KF96 Store the SNAP Solution at 4 C Store all other kit components at room temperature 1 x 96 extractions 5 x 96 extractions Catalogue Number 7460300100 7460300200 Buffer EL conc 2x30 ml 8 x 30 ml Lysis Solution TR 60 ml 260 ml SNAP Solution 2x 1 1 ml 10 5 ml 80 ml 2x 125 ml Wash Buffer R1 final volume 160 ml final volume 2 x 250 ml Wash Buffer R2 40 ml final volume 200 ml 4x 50 ml final volume 4 x 250 ml Elution Buffer R 15 ml 60 ml 2 0 ml Deep Well Plate 4 20 KF96 TipComb for 1 5 DW magnets 200 ul Elution Plate 2 10 1 5 ml Receiver Tubes 2x50 10 x 50 Manual 1 1 Initial steps Add 30 ml Buffer EL to 970 ml pure Add 30 ml Buffer EL to 970 ml pure water 2x Add 80 ml of 96 100 ethanol to the bottle Wash Buffer R1 Add 160 ml of 96 100 ethanol to the bottle Wash Buffer R2 Mix thoroughly and always keep the bottles firmly closed water 8x Add 125 ml of 96 100 eth
22. l time RT PCR or RT 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 RNA 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 22 InviMag Universal RNA Mini Kit KF96 0515 Appendix KingFisher Software Version 2 6 and 3 1 The KingFisher Software 2 6 2 was used for the creation of the KingFisher 96 protocols whereas the Software 3 1 was used for creation of KingFisher Flex 96 run files The user can either transfer the protocol onto the workstation or run the the protocol directly from the software Be aware that directly run protocols are not stored in the workstation memory If you don t have the correct KingFisher software installed on your computer please call your local ThermoFisher distributor for an update Note Please keep in mind that software version 2 6 and 3 1 are not compatible It is not possible to run a procotol created in version 2 6 under version 3 1 and vice versa Minimal PC Requirements for KingFisher Software 2 6 and 3 1 PC requirements Interface Serial communication port via
23. n of Ethanol The RNA bound to the simultaneously added magnetic particles is separated from the solution by magnetic rods controlled by the KingFisher instrument Subsequent to the washing steps of the particle bound nucleic acids the RNA is eluted in Elution Buffer R Due to the high purity the eluted RNA is ready to use in a broad panel of downstream applications RNA dot blots cDNA transcription Real time PCR quantitative RT PCR like TaqMan und LightCycler technologies Array technologies oO 0 0 The results from downstream applications 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 For the isolation of RNA from a single sample STRATEC Molecular offers the Invisorb Spin Cell RNA Mini Kit or Invisorb Spin Tissue RNA Mini Kit for use on a centrifuge see Ordering information page 24 For further information please contact phone 49 0 30 9489 2901 2910 in Germany and from foreign countries phone 49 0 30 9489 2907 2903 or your local distributor The PCR process is covered by US Patents 4 683 195 and 4 683 202 and foreign equivalents owned by Hoffmann La Roche AG InviMag Universal RNA Mini Kit KF96 0515 Sampling and storage of starting material Tissues cells and blood may be used fresh or frozen stored at 80 C STRATEC Molecular will be released of it
24. n to execute the run At the end of the extraction protocol the isolated RNA is loacted in the Elution Plate In case of long term storage we recommend to to seal the plate or transfer the isolated RNAs into a 1 5 ml reaction tubes and freeze them at 80 TC 16 InviMag Universal RNA Mini Kit KF96 0515 ll Extraction steps automatically performed on the KingFisher system Binding of the DNA Automatically sample mixing for 5 min SNAP separation SNAP transfer to Washing Plate 1 First Washing Automatically sample mixing for 2 min SNAP separation SNAP transfer to Washing Plate 2 Second Washing Automatically sample mixing for 1 min SNAP separation SNAP transfer to Washing Plate 3 Third Washing and Drying Automatically sample mixing for 1 min SNAP separation Drying the SNAP outsight Washing Plate 3 for 7 minutes SNAP transfer to Elution Plate Elution of the DNA Automatically sample mixing for 10 minutes SNAP separation and removal into Washing Plate_3 disposal The extracted DNA can now be transferred into 1 5 ml reaction tubes Important Notes After the run the Elution Plate contains the extracted RNA Store the RNA under adequate conditions We recommend to transfer the extracted RNA into an 1 5 ml reaction tube for further storage and freeze the DNA at 20 C Alternatively the Elution Plate can be sealed and stored at 80 If the extracted RNA contains carryover of magnetic particles transfer the RNA into a
25. ntrifuge the suspension for 1 min at maximum speed e g 13 000 rpm on a table centrifuge Carefully transfer the supernatant to the prepared Binding Plate without disturbing the pellet Table 1 Lysis Solution TR 1MDTT Number of pelleted cells 500 ul 5 ul up to 1x10 cells b Monolayer cells Add Lysis Solution TR with DTT see tab 2 DTT is not supplied within the kit to the monolayer cells Collect the cell lysate with a rubber policeman Mix thoroughly by pipetting up and down until no cell clumps are visible Incubate for 2 min before proceeding with the next step Centrifuge the suspension for 1 min at maximum speed e g 13 000 rpm on a table centrifuge Carefully transfer the supernatant to the prepared Binding Plate without disturbing the pellet Table 2 Lysis Solution TR 1MDTT Size of the culture vessels 500 ul 5 ul 96 24 12 well plates 13 InviMag Universal RNA Mini Kit KF96 0515 Protocol 2 Extraction of RNA from fresh or frozen tissues Please read the instructions carefully and conduct the prepared procedure The disruption procedure the breakage of intercellular matrix like cells walls organelles and plasma membranes is necessary to release the nucleic acids contained in the cell thus inefficient disruption decreases the RNA yield Different samples require different methods to achieve complete disruption The homogenization means the reduction of the viscosity of the lysate
26. ood Please read the instructions carefully and conduct the prepared procedure Note The centrifugation steps were performed with the Centrifuge 5415 D from Eppendorf The incicated rom amounts are referring to this centrifuge 1 Lysis of Erythrocytes Add 10 ml of cold 4 C prepared Buffer EL to 0 5 1 5 ml of whole blood max 1 x 10 leukocytes in a 15 ml tube e g 15 ml Falcon Tube not provided Mix shortly but completely by vortexing Incubate on ice for 15 min Mix briefly by vortexing two times during incubation Note The cloudy suspension becomes translucent during incubation indicating lysis of erythrocytes If necessary the incubation time can be extended to 20 min Centrifuge at 1 700 x g 3 000 rpm for 5 min at 4 C carefully remove and discard the supernatant completely without disturbing the visible white leukoytes pellet Note Leukocytes will form a pellet after centrifugation Ensure that the supernatant is completely removed by aspiration Trace amounts of erythrocytes which give the pellet the red tint will be eliminated by following washing steps Important If fresh blood samples up to 4 h after taking the sample are used extend the lysis time of erythrocytes to 30 min that the total lysis time is at least 45 min Please keep in mind that up to 1 5 ml whole blood can be processed If the expected amount of leukocytes is more than 1x10 reduce the starting volume of the blood sample Add
27. pure water 8x Add 125 ml of 96 100 ethanol to each bottle Wash Buffer R1 Add 200 ml of 96 100 ethanol to each bottle Wash Buffer R2 Mix thoroughly and always keep the bottles firmly closed Reagents and equipment to be supplied by user Measuring cylinder 250 ml Pipette and pipette tips Disposable gloves ddH O Vortexer 96 100 Ethanol oO000 0 0 10 InviMag Universal RNA Mini Kit KF96 0515 Product validation Figure 1 The quality and yield of the InviMag Universal RNA Mini Kit KF96 was ensured by multiple testing Figure 1 shows the result of a typical RNA extraction of 10 20 mg different tissues using two types of homogenizers Heart Liver Brain Gut Lu ng I I I MBBGGBBGGBBGGBBGCGCBBGG M Marker Generuler Fermentas B Bulltet Blender NEXT G Gyrator STRATEC Molecular 10 ul sample respectively was used for gel analysis M S S2 M M Marker Generuler Fermentas 3 5 ul S Sample Figure 2 shows the typical result of a RNA preparation of leukocytes derived from 1 5 ml whole blood using with the InviMag Universal RNA Mini Kit KF96 M Marker Generuler Fermentas 10 ul sample respectively was for gel analysis Figure 3 shows a common result of a RNA preparation of two cell lines using different amount of cells performed with the InviMag Universal RNA Mini Kit KF96 11 InviMag Universal RNA Mini
28. quid spillage refer to Safety Information see page 6 Do not use damaged kit components since their use may lead to poor kit performance o Always change pipet tips between liquid transfers To avoid cross contamination we recommend the use of aerosol barrier pipet tips o All centrifugation steps are carried out at room temperature When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles Discard gloves if they get 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 o This kit should only be used by trained personnel O O TOO InviMag Universal RNA Mini Kit KF96 0515 Preparing reagents and buffers Before starting a run bring all reagents to room temperature When necessary gently mix and redissolve any precipitates by warming up to 30 C Swirl gently to avoid foaming Lysis Solution TR and Elution Buffer R are ready to use 1x 96 extractions Add 30 ml Buffer EL to 970 ml pure water 2x Add 80 ml of 96 100 ethanol to the bottle Wash Buffer R1 Add 160 ml of 96 100 ethanol to the bottle Wash Buffer R2 Mix thoroughly and always keep the bottles firmly closed 5x 96 extractions Add 30 ml Buffer EL to 970 ml
29. rovide 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 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 Universal RNA 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 di
30. s 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 Universal RNA Mini Kit KF96 procedure comprises the following steps o Lysis or homogenization of the material in Lysis Solution TR o Binding of the RNA to magnetic beads and removal of DNA by the contained carrier o Washing steps and elimination of ethanol o Elution of RNA After lysis and removal of DNA the RNA binds to the magnetic beads whereas contaminations and enzyme inhibitors are efficiently removed during the following three washing steps Highly purified RNA is eluted in Elution Buffer R This manual contains 3 protocols Yield and quality of RNA The amount of purified RNA in the InviMag Universal RNA Mini Kit KF96 procedure depends on the type and condition of the cells or tissues used Yield and quality of isolated RNA is suitable for any downstream processing 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 li
31. s throughout the procedure these tubes are generally RNase free Keep isolated RNA on ice o Do not use kit components from other kits with the kit you are currently using unless the lot numbers are identical o Tominimize the risk of infections from potentially infectious material we recommend working under laminar air flow until the samples are lysed oO000 0 O O This kit should only be used by trained personnel Storage of RNA Purified RNA can be stored at 80 C and is stable for several years at this condition Quantification of RNA The concentration of RNA should be determinate by measuring the absorbance at 260 nm A260 in a spectrophotometer Readings should be greater than 0 10 to ensure significance An absorbance of 1 unit at 260 nm corresponds to 40 ug of RNA per ml This relation is valid only for measurements at neutral pH The ratio between absorbance values at 260 nm and 280 nm gives an estimate of RNA purity see below When measuring RNA samples make sure that cuvettes are RNase free esp if the RNA is to be recovered after spectrophotometry This can be accomplished by washing cuvettes 24 InviMag Universal RNA Mini Kit KF96 0515 with 0 1 NaOH 1 mM EDTA followed by washing with RNase free water Use the buffer in which the RNA is diluted for calibration of the spectroohotometer An example of the calculation involved in RNA quantification o Volume of RNA sample 100 ul o Dilution 20 ul of RN
32. scarding the bottles in order to avoid any injuries STRATEC Molecular has not tested the liquid waste generated by the InviMag Universal RNA Mini Kit KF96 procedures for residual risk materials Therefore liquid waste should be handled and treated accordingly to local safety regulations European Community risk and safety phrases for the components of the InviMag Universal RNA Mini Kit KF96 to which they apply are listed below as follows Lysis Solution TR and Wash Buffer R1 contain a chaotropic salt which is an irritant Wash Buffer R1 and Elution Buffer R contain DEPC treated dd H O DEPC is inactivated by autoclaving for 20 minutes at 121 C Lysis Solution TR Wash Buffer R1 l warning 9 warning contains guanidine thiocyanate contains guanidine thiocyanate H302 312 332 412 EUH032 P273 H302 312 332 412 EUH032 P273 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 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 1 800 535 5053 7 InviMag Universal RNA Mini Kit KF96 0515 Product characteristic of the InviMag Universal RNA Mini Kit KF96 The InviMag Universal RNA Mini Kit KF96 procedure is the ideal tool for efficient extraction and
33. t No Release beads No Mixing heating parameters Heating during mixing No Mixing time hh mm ss 00 05 00 Mixing speed Slow End of step Postmix No Collect count 5 Collect time s 2 WASHING PLATE 1 Beginning of step Precollect No Release time hh mm ss 00 00 30 Release speed Medium Mixing heating parameters Heating during mixing No Mixing time hh mm ss 00 02 00 Mixing speed Medium End of step Postmix No Collect count 3 Collect time s 2 WASHING PLATE 2 Beginning of step Precollect No Release time hh mm ss 00 00 30 Release speed Medium Mixing heating parameters Heating during mixing No Mixing time hh mm ss 00 01 00 Mixing speed Medium InviMag RNA Universal Kit KF96 Kfflex96 Vers 0315 End of step Postmix No Collect count 3 Collect time s 2 WASHING PLATE 3 Beginning of step Precollect No Release time hh mm ss 00 00 30 Release speed Medium Mixing heating parameters Heating during mixing No Mixing time hh mm ss 00 01 00 Mixing speed Medium End of step Postmix No Collect count 3 Collect time s 2 21 DRYING Plate Washing Plate 3 Dry time hh mm ss 00 07 00 Tip position Outside well tube ELUTION Beginning of step Precollect No Release time hh mm ss 00 00 30 Release speed Medium Mixing heating parameters Heating during mixing No Mixing time hh mm ss 00 10 00 Mixing speed Slow End of s
34. tep Postmix No Collect count 5 Collect time s 2 REMOVE BEADS Plate Washing Plate 3 Release time hh mm ss 00 00 30 Release speed Fast InviMag Universal RNA Mini Kit KF96 0515 Troubleshooting Problem Probable cause Comments and suggestions low amount of extracted RNA insufficient lysis incomplete elution low amount of SNAP Solution increase lyses time but prevent too long lyses times because this also decrease yield reduce amount of starting material take higher volume of Elution Buffer R be sure you pipet the Elution Buffer R with the right amount to the right position mix SNAP Solution thoroughly before use low concentration of extracted RNA too much Elution Buffer incorrect storage of starting material incorrect Wash Buffers elute the RNA with lower volume of Elution Buffer R ensure that the storage of starting material was correctly avoid repeated thawing of the material make sure that the correct amount of ethanol is added to the Wash Buffers and store them correctly degraded RNA incorrect storage of starting material old material ensure that the storage of starting material is correctly avoid thawing of the material ensure that the starting material is fresh or stored under appropriate condition for long time storage at 80 C avoid thawing of the material RNA does not perform well in downstream applications e g rea
35. use to ensure that it is RNase free Glassware should be cleaned with detergent thoroughly rinsed and oven baked at 240 C for four or more hours before use Autoclaving alone will not completely inactivate many RNases Oven baking will both inactivate RNases and ensure that no other nucleic acids such as Plasmid DNA are present on the surface of the glassware You can also clean glassware with 0 1 DEPC diethyl pyrocarbonate The glassware has to stand 12 hours at 37 C and then autoclave or heat to 100 C for 15 min to remove residual DEPC o Electrophoresis tanks should be cleaned with detergent solution e g 0 5 SDS thoroughly rinsed with RNase free water and then rinsed with ethanol and allowed to dry o Non disposable plastic ware should be treated before use to ensure that it is RNase free Plastic ware should be thoroughly rinsed with 0 1 M NaOH 1 mM EDTA followed by RNase free water You can also take chloroform resistant plastic ware rinsed with chloroform to inactivate RNases o All buffers must be prepared from DEPC treated RNase free ddH20 When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles Change gloves frequently and keep tubes closed All centrifugation steps are carried out at room temperature To avoid cross contamination cavity seams shouldn t be moisted with fluid Reduce the preparation time as much as possible Use only sterile disposable polypropylene tube
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