InviTrap RNA Tissue HTS 96 Kit/ C User manual
Contents
1. Gi InviTrap RNA Tissue stratecee molecular User manual HTS 96 Kit C for purification of total RNA from 1 10 mg fresh or frozen tissue sample without DNase digestion ina 96 well format using a centrifuge CE 7062300x00 tsal STRATEC Molecular GmbH D 13125 Berlin aa Instruction for InviTrap RNA Tissue HTS 96 Kit C The InviTrap RNA Tissue HTS 96 Kit C is designed for isolation of high quality total RNA from 96 samples of fresh or frozen tissue sample No enzymatic step e g DNase digestion to remove contaminating genomic DNA is necessary Due to the high purity the isolated total RNA is ready to use for a broad panel of downstream applications The InviTrap RNA Tissue HTS 96 Kit C is optimized for use on a centrifuge Compliance with EU Directive 98 79 EC on in vitro medical devices Not for in vitro diagnostic use in countries where the EU Directive 98 79 EC on in vitro medical devices is not recognized Trademarks InviTrap Invisorb Eppendorf Sigma KNF VACUUBRAND TaqMan 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 InviTrap and Invisorb are registered trademark2. RNA Tissue HTS 96 Kit C 2 x 96 preps 7062300200 InviTrap RNA Tissue HTS 96 Kit C 4 x 96 preps 7062300300 InviTrap RNA Tissue HTS 96 Kit C 24 x 96 preps 7062300400 InviMag RNA Universal mini Kit KF96 1 x 96 preps 7460300100 InviMag RNA Universal mini Kit KF96 5 x 96 prep 7460300200 InviTrap Spin Universal RNA Mini Kit 10 preps 1060100900 InviTrap Spin Universal RNA Mini Kit 50 preps 1060100200 InviTrap Spin Universal RNA Mini Kit 250 preps 1060100300 11 InviTrap RNA Tissue HTS 96 Kit C 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 1C5kC 05 2015
3. Elution Plate L 2 4 24 RNA Binding Plate A 2 4 6x4 Plate Lid 2 4 24 Manual 1 1 1 Initial steps add 84 ml 96 100 add 140 ml 96 100 I add 420 ml 96 1009 ethanol to the bottle Binding Buffer R add 60 ml 96 100 ethanol to the bottle Wash Buffer R1 add 120 ml 96 100 ethanol to the bottle Wash Buffer R2 ethanol to each bottle Binding Buffer R add 120 ml 96 100 ethanol to each bottle Wash Buffer R1 add 120 ml 96 100 ethanol to each bottle Wash Buffer R2 ethanol to each bottle Binding Buffer R add 350 ml 96 100 ethanol to each bottle Wash Buffer R1 add 720 ml 96 100 ethanol to each bottle Wash Buffer R2 InviTrap RNA Tissue HTS 96 Kit C 0515 Symbols Tm Lot number Catalogue number Expiry date Consult operating instructions Temperature limitation Do not reuse SHI Storage The InviTrap RNA Tissue HTS 96 Kit C should be stored dry at room temperature It is stable for at least 12 months under these conditions Room temperature RT is defined as range from 15 30 C Before every use make sure that all components have room temperature If there are any precipitates within the provided solutions solve these precipitates by warming carefully Quality control and product warranty STRATEC Molecular warrants the correct function of the InviTrap RNA Tissue HTS 96 Kit C for applications as described in this manual Purchaser must d
4. high yields an appropriate sample storage and guick operation under the rules for RNA operation is essential The purified RNA can be used for in vitro diagnostic analysis The InviTrap RNA Tissue HTS 96 Kit C is optimized for use with a centrifuge THE PRODUCT IS INDENTED FOR USE BY PROFESSIONAL USERS 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 The kit is in compliance with EU Directive 98 79 EC on in vitro medical devices But it is not for in vitro diagnostic use in countries where the EU Directive 98 79 EC on in vitro medical devices is not recognized Product use limitation The kit is neither validated for the isolation of total RNA from serum plasma bacteria or yeast cells nor for viruses The performance of the kit in isolating and purifying total RNA from fecal samples has not been evaluated The kit was not tested on its ability to desalinate RNA or for RNA purification from enzymatic reactions
5. like Proteinase digestion RNA ligation or labeling reactions The included chemicals are for one time use plates can be used several times if not all wells were used in first run and unused wells were sealed during the process and no contamination occurred When changing the starting material or the flow trace no guarantee in operability is issued The user is responsible to validate the performance of the STRATEC Molecular kits for any particular use since the performance characteristics of our kits have not been validated for any specific application STRATEC Molecular kits may be used in clinical diagnostic laboratory systems after the laboratory has validated the complete diagnostic system as required by CLIA 88 regulations in the U S or equivalents in other countries All products sold by STRATEC Molecular are subjected to extensive quality control procedures according to ISO 9001 2000 and ISO EN 13485 2003 and are warranted to perform as described when used correctly Any problems should be reported immediately The chemicals and plastic parts are for laboratory use only they must be stored in the laboratory and must not use for purposes other than intended The kit contents are unfit for consumption 5 InviTrap RNA Tissue HTS 96 Kit C 0515 Safety information When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles Avoid skin contact Heed the legal requirements for working w
6. 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 6 InviTrap RNA Tissue HTS 96 Kit C 0515 Product characteristic of the InviTrap RNA Tissue HTS 96 Kit C 1 mg 10 mg fresh or e g 30 ug total RNA from mouse liver about 50 min frozen tissue sample tissue The InviTrap RNA Tissue HTS 96 Kit C is designed to isolate high yield of cellular total RNA from 1 mg up to 10 mg fresh or frozen tissue sample in a 96 well format No enzymatic step DNase digestion to remove contaminated genomic DNA is necessary Special buffer conditions guarantee an efficient lysis of the starting material and an inactivation of endogenous RNases Genomic DNA contaminations are almost completely separated from the total RNA by binding to specially optimized mineralic carrier particles Downstream Application o RT PCR o DDRT PCR o Real time PCR o Northern Blot Reagents and equipment to be supplied by user o Multichannel pipet with tips Reagents reservoirs for multichannel pipets Centrifuge for microplates e g Eppendorf 5804 5804 R or 5810 5810 R centrifuge with deepwell plate rotor Ethanol 96 100 dd H2O Liquid N2 Mill for homogenization of starting material under liquid N2 O O O O CO 0 The PCR method is covered by U S Patents 4 683 195 and 4 683 202 owned by Hoffmann LaRoche Inc The purchase of the InviTrap RNA Tiss
7. etermine the suitability of the Product for its particular use Should any Product fail to perform the applications as described in the manual STRATEC Molecular will check the lot and if STRATEC Molecular investigates a problem in the lot STRATEC Molecular will replace the Product free of charge STRATEC Molecular reserves the right to change alter or modify any Product to enhance its performance and design at any time In accordance with STRATEC Molecular s ISO 9001 2000 and ISO EN 13485 certified Ouality Management System the performance of all components of the InviTrap RNA Tissue HTS 96 Kit C 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 InviTrap RNA Tissue HTS 96 Kit C 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 4 InviTrap RNA Tissue HTS 96 Kit C 0515 Intended use The InviTrap RNA Tissue HTS 96 Kit C has been designed to isolate high yield of cellular total RNA from 1 mg up to 10 mg fresh or frozen tissue sample in a 96 well format For reproducible and
8. ith 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 kit and kit component If the 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 InviTrap RNA Tissue HTS 96 Kit C procedures for residual infectious materials Contamination of the liquid waste with residual infectious materials is highly unlikely but cannot be exclude completely Therefore liquid waste must be considered infectious and be handled and discarded according to local safety regulation Below are listed the European Community risk and safety phrases for the components of the InviTrap RNA Tissue HTS 96 Kit C to which they apply Lysis Solution DC Wash Buffer R1 and Lysis Solution D contain a chaotropic salt which is an irritant Always wear gloves while handling these reagents and avoid any skin contact Lysis Solution DC Wash Buffer R1 warning danger H302 312 332 412 EUH 032 P273 H302 312 332 412 EUHO32 P273 H302 Harmful if swallowed H312 Harmful in contact with skin H332 Harmful if inhaled H412 Harmful to aguatic life with long lasting effects EUH032 Contact with acids liberates very toxic gas P273 Avoid release to the environment
9. ll of the RNA Binding Plate C Close the RNA Binding Plate Awith the Plate Lid Load the whole block RNA Binding Plate A 2 ml Collection Plate into the holder and place the whole assembly in the rotor bucket Centrifuge at 4 000 rom for 5 min at RT Take the RNA Binding Plate out of the centrifuge open the Plate Lid ad place the plate on a clean paper towel 6 Removing of ethanol Empty the 2 ml Collection Plate and dry its upper side with paper Place the RNA Binding Plate Aon top of the 2 ml Collection Plate and close it with the Plate Lid Load the whole block RNA Binding Plate A 2 ml Collection Plate into the holder and place the whole assembly in the rotor bucket of the centrifuge Centrifuge at maximum speed for at least 15 min at RT to eliminate any traces of ethanol Take the RNA Binding Plate A 2 ml Collection Plate out of the centrifuge remove the Plate Lid and place the plate on a clean paper towel Discard the 2 ml Collection Plate 9 InviTrap RNA Tissue HTS 96 Kit C 0515 7 Elution of the total RNA Place the RNA Binding Plate Aon top of a RNase free Elution Plate L Add 80 ul Elution Buffer R RNase free directly onto the membrane in each well and incubate for 5 min Close the RNA Binding Plate A with the Plate Lid and place the whole block RNA Binding Plate A Elution Plate L in the rotor bucket of the centrifuge Centrifuge for 5 min at 4000 rpm Take the RNA Binding Plate A and the Elution Plate L out of the cen
10. n downstream applications e g RT PCR Ethanol carryover during elution Salt carryover during elution Increase g force or centrifugation time in step 8 Ensure that Wash Buffer R1 and R2 are at room temperature Check up Wash Buffer R1 and R2 for salt precipitates If there are any precipitates solve these precipitates by careful warming 10 InviTrap RNA Tissue HTS 96 Kit C 0515 Appendix General notes and safety recommendations 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 gualitative and guantitative results in RNA preparations contaminations with exogenous RNases have to be reduced as much as possible in accordance with the following recommendations o OO C00O0 Always wear latex or vinyl gloves while handling reagents and RNA samples to prevent RNase contaminations from surface of the skin or from dusty laboratory equipment Change gloves frequently and keep tubes closed Keep isolated RNA on ice when aliguots are pipetted for downstream applications Reduce preparation time as much as possible Use only sterile disposable polypropylene tubes throughout the procedure these tubes are generally RNase free Non disposable plasticware should be treated before use to ensure that it is RNase free Plasticware should be thoro
11. n Plate very carefully Avoid carryover of unlysed material 3 Adjust RNA binding conditions Add 450 ul Binding Buffer R to each well of the collection plate and mix the solution very well by pipetting several times using a multichannel pipet Place the RNA Binding Plate Aon the top of a 2 ml Collection Plate Transfer the samples completely into each well of the RNA Binding Plate A Close the RNA Binding Plate A with the Plate Lid and incubate for 1 min at RT Load the whole block RNA Binding Plate A 2 ml Collection Plate into the holder and place the whole assembly in the rotor bucket Centrifuge at 4 000 rom for 5 min at RT Take the RNA Binding Plate A 2 ml Collection Plate out of the centrifuge Remove the Plate Lid and discard the filtrate Place the RNA Binding Plate Aback to the top of the 2 ml Collection Plate 4 First washing of the RNA Binding Plate A Add 500 ul Wash Buffer R1 to each well of the RNA Binding Plate C Close the RNA Binding Plate Awith the Plate Lid Load the whole block RNA Binding Plate C 2 ml Collection Plate into the holder and place the whole assembly in the rotor bucket Centrifuge at 4000 rpm for 5 min at RT Take the RNA Binding Plate out of the centrifuge open the Plate Lid and place the plate on a clean paper towel Discard the filtrate and place the RNA Binding Plate Aback to the top of the 2 ml Collection Plate 5 Second washing of the RNA Binding Plate C Add 700 ul Wash Buffer R2 to each we
12. s 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 1 InviTrap RNA Tissue HTS 96 Kit C 0515 Contents Kit content of InviTrap RNA Tissue HTS 96 Kit C Symbols Storage Quality control Intended use Product use limitation Safety information Product characteristic of the InviTrap RNA Tissue HTS 96 Kit C Equipment and reagents to be supplied by user Scheme Oo ON NO aA a FP BP A OQ Protocol RNA extraction from 1 mg 10 mg tissue material mh o Troubleshooting Appendix h l adil a Ordering information 2 InviTrap RNA Tissue HTS 96 Kit C 0515 Kit Content of InviTrap RNA Tissue HTS 96 Kit C Store all kit components at room temperature 2 x 96 RNA preps 4x 96 RNA preps 24 x 96 RNA preps Catalogue No 7062300200 7062300300 7062300400 Lysis Solution DCT 120 ml 220 ml 2 x 650 ml 36 ml 60 ml 2 x 180 ml Binding Buffer R final volume 120 ml final vol 1 x 200 ml final vol 2 x 600 ml Elution Buffer R 30 ml 60 ml 200 ml Wash Buffer R1 60 ml final volume 120 ml 120 ml final volume 240 ml 2 x 350 ml final vol 2 x 700 ml Wash Buffer R2 30 ml final vol 150 ml 2x 30 ml final vol 2 x 150 ml 2 x 180 ml final vol 2 x 900 ml 2 0 ml Collection Plate 2x3 3x4 18x4
13. trifuge very carefully in order to avoid cross contaminations with adherent fluid Discard the RNA Binding Plate Awith the Plate Lid Take the Elution Plate L with the eluted total RNA and cover the plate with sealing foil or the cover Store at 80 C Troubleshooting Problem probable cause Comments and suggestions Clogged Plate Filter insufficient disruption or homogenisation After lysis spin lysate to pellet debris and continue with the protocol using the supernatant Reduce amount of starting material Little or no total RNA eluted insufficient disruption or homogenisation incomplete elution Reduce amount of starting material Overloading reduces yield Prolong the incubation time with Elution Buffer R to 5 10 min or repeat elution step once again DNA contamination to much material Total RNA degraded RNA source inappropriately handeled or stored RNase contaminations of solutions receiver tubes etc Reduce amount of starting material DNase digests the eluate containing the total RNA Ensure that the starting material is frozen immediately in liquid Nz and is stored continuously at 80 C Avoid thawing of the material Ensure that the protocol especially the first steps has been performed quickly Use sterile RNase free filter tips Before every preparation clean up the pipets the devices and the working place Always wear gloves Total RNA does not perform well i
14. ue HTS 96 Kit C cannot be construed as an authorization or implicit licence to practice PCR under any patents held by Hoffmann LaRoche Inc 7 InviTrap RNA Tissue HTS 96 Kit C 0515 Scheme of the InviTrap RNA Tissue HTS 96 Kit C 1 Lysis NM Adjust RNA binding conditions ao Binding of total RNA to the RNA Binding Plate A 4 Wash steps to remove contaminants 1 x Wash Buffer R1 1 x Wash Buffer R2 5 Elution of total RNA Please work quickly and perform all extraction steps at room temperature RT 8 InviTrap RNA Tissue HTS 96 Kit C 0515 Protocol RNA extraction from 1 mg 10 mg tissue material Important Shake Lysis Solution DCT gently before use Tissue sample can be stored under Lysis Solution DCT at 20 TC for several month 1 Tissue disruption Grind sample under liquid nitrogen thoroughly to a fine powder using a mortar and pestle or using commercial available homogenizers e g mills Incomplete grinding of the tissue will lead to reduced total RNA yield Transfer the tissue powder max 10 mg into the cavities of the 2 ml Collection Plate 2 Lysis of starting material Important Before every use mix Lysis Solution DCT thoroughly by inverting the bottle 5 10 times Add 0 5 ml Lysis Solution DCT to each cavity Mix several times by pippetting up and down Incubate for 15 min at RT Centrifuge at maximum speed for 5 min Transfer 450 ul from the supernatant into a new 2 ml Collectio
15. ughly rinsed with 0 1 M NaOH 1 mM EDTA followed by RNase free water You can also take chloroform resistant plasticware rinsed with chloroform to inactivate RNases All glassware should be treated before 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 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 All buffers must be prepared from DEPC treated RNase free JddH O Avoid handling bacterial cultures cell cultures or other biological sources of RNases in the same lab where the RNA purification has to be carried out Do not use equipment glassware and plasticware employed for other applications which might introduce RNase contaminations in the RNA isolation Storage of RNA Purified viral RNA can be stored at 80 C and is stable for several years at this condition Ordering information Product Package size Catalogue No InviTrap
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