NucleoSpin® 8 RNA - MACHEREY
Contents
1. Splattering of eluate Reduce the vacuum strength during the elution step Alternatively a Round well Block or Rack of Tube Strips see ordering information can be used for collecting the eluate if a Cross contamination higher vacuum strength is required during the elution Transfer of sample solution to the NucleoSpin RNA Binding Strips Be sure that no liquid drops out of the tips while moving the tips above the binding plate 6 2 Ordering information Product REF Pack of NucleoSpin 8 RNA 740698 12x 8 preps 740698 5 60 x 8 preps NucleoSpin 8 RNA Core Kit 740465 4 48 x 8 preps NucleoSpin 96 RNA 740709 2 2 x 96 preps 740709 4 4x 96 preps 740709 24 24 x 96 preps NucleoSpin 96 RNA Core Kit 740466 4 4x 96 preps NucleoSpin RNA Filter Strips 740699 12F 12 740699 60F 60 Buffer RA1 740961 55 500 mL Buffer RA4 Concentrate 740960 300 mL for 1 L Buffer RA4 TCEP 740395 107 107g MN Square well Block 740476 4 740476 24 24 Round well Block Low 740487 4 sets set consists of 1 Round well Block Low and 740487 24 24 sets Self adhering PE Foil MACHEREY NAGEL 04 2014 Rev 05 35 Total RNA isolation Product REF Pack of Round well Block with Cap Strips 740475 4 sets set consists of 1 Round well Block 12 740475 24 24 sets Cap Strips Rack of Tube Strips with Cap Strips 740477 4 sets set consists of 1 Rack 12 Tube Strips with 8 740477 24 24 sets tubes each and 12 Cap Strips Elution Pl2. Full processing under vacuum enables complete automation without the need of centrifugation steps for drying or elution However a final elution step by centrifugation is recommended in order to achieve higher concentrated DNA eluates The risk of cross contamination is reduced by optimized vacuum settings during the elution step and by the improved shape of the outlets of the NucleoSpin RNA Binding Strips Drying of the NucleoSpin RNA Binding Strips only under vacuum is sufficient as the bottom of the plate is protected by the MN Wash Plate during the washing steps As a result it is recommended integrating the MN Wash Plate into the automated procedure The MN Frame see ordering information can be used to place the disposable MN Wash Plate inside the vacuum chamber This also reduces the risk of cross contamination as common metal adaptors tend to get contaminated by gDNA Thorough cleaning of the 10 MACHEREY NAGEL 04 2014 Rev 05 Total RNA isolation vacuum chamber is recommended after each run to prevent DNA containing aerosols from forming 2 7 Sample torage and homogenization RNA is not protected against digestion until the sample material is flash frozen or disrupted in the presence of RNase inhibiting or denaturing agents Therefore it is important that samples are either immediately frozen and stored at 70 C placed in a RNA stabilizing reagent not included in the kit or processed as soon as harvested Aft
3. Total RNA isolation User manual NucleoSpin 8 RNA NucleoSpin 8 RNA Core Kit April 2014 Rev 05 MACHEREY NAGEL MN www mn net com Total RNA isolation Table of contents 1 Components 1 1 Kit contents 1 2 Reagents to be supplied by user 5 2 Product description 6 2 1 The basic principle 6 2 2 Kit specifications 6 2 3 Yield and quality of total RNA 6 2 4 Required hardware 8 2 5 Recommended accessories for use of the NucleoSpin 8 RNA Core Kit 9 2 6 Automated processing on robotic platforms 10 2 7 Sample torage and homogenization 11 2 8 Elution of pure total RNA 13 3 Storage conditions and preparation of working solutions 14 4 Safety instructions 16 5 Protocols 18 5 1 NucleoSpin 8 RNA vacuum processing 18 5 2 NucleoSpin 8 RNA centrifuge processing 25 5 3 Automated purification using common laboratory automation workstations 30 5 4 Clean up of total RNA 32 6 Appendix 33 6 1 Troubleshooting 33 6 2 Ordering information 35 6 3 Product use restriction warranty 36 MACHEREY NAGEL 04 2014 Rev 05 3 Total RNA isolation 1 Components 1 1 Kit contents NucleoSpin 8 RNA 12 x 8 preps 60 x 8 preps REF 740698 740698 5 Lysis Buffer RA1 60 mL 250 ml Wash Buffer RA2 60 mL 360 ml Wash Buffer RAS 50 mL 2x 100 ml Concentrate Wash Buffer De 65 mL 3 x 65 ml Concenitrate Reaction Buffer for rDNase 30 mL 2x60 ml rDNase RNase free E lyophilized 2 vials 10 vials RNase free H O 3
4. Add 800 uL Buffer RA3 to each well of the NucleoSpin RNA Binding Strips and centrifuge for or 2 min at 5 600 6 000 x g Empty MN Square well Block Add 500 uL Buffer RA4 to each well of the NucleoSpin RNA Binding Strips and centrifuge for or 10 min at 5 600 6 000 x g Empty MN Square well Block 10 Dry NucleoSpin RNA Binding Strips Residual wash buffer from the NucleoSpin RNA Binding Strips is removed by the prolonged centrifugation time of 10 min after adding Wash Buffer RA4 as described in step 8 This prolonged time is necessary to eliminate traces of ethanol Note The ethanol in Buffer RA4 inhibits enzymatic reactions and has to be removed completely before eluting DNA Elute RNA For elution place Column Holder C with the NucleoSpin RNA Binding Strips onto the Rack of Tube Strips and pipette 75 yl RNase free H O directly to the bottom of each well 75 uL are recommended 50 130 uL are possible see section 2 8 Make sure that all of the water gets into contact with the silica membrane and that tne membrane is completely wetted Incubate for 2 min at room temperature and for 3 min at 5 600 6 000 x g Alternatively elution in a MN Square well Block or Round well Block see ordering information is possible Note The Elution Plate U bottom is not suitable for use in a centrifuge MACHEREY NAGEL 04 2014 Rev 05 29 Automated purification using laboratory automation workstations 5 3 Automated puri
5. Total RNA from 5 x 10 HeLa cells Empty control well next to a well containing cells a 200 bp product of the GAPDH gene b lt lt lt lt lt lt lt lt Primers MACHEREY NAGEL 04 2014 Rev 05 13 Total RNA isolation 3 Storage conditions and preparation of working solutions Attention Buffers RA1 and RA2 contain chaotropic salts Wear gloves and goggles CAUTION Buffers RA1 and RA2 contain guanidinium thiocyanate which can form highly reactive compounds when combined with bleach sodium hypochlorite DO NOT add bleach or acidic solutions directly to the sample preparation waste Storage conditions Store lyophilized RNase free rDNase at 4 C on arrival stable for at least one year All other components of the NucleoSpin 8 RNA kit should be stored at room temperature and are stable for at least one year Storage at lower temperatures may cause precipitation of salts If a salt precipitate is observed incubate the bottle at 30 40 C for some minutes and mix well until all of the precipitate is redissolved Before starting with any NucleoSpin 8 RNA protocol prepare the following Reconstitute RNase free rDNase Add 540 uL RNase free H O to the rDNase vial and incubate for 1 min at room temperature Gently swirl the vial to completely dissolve the rDNase In case of not processing a whole 96 well plate dispense the reconstituted rDNase solution into aliquots and store at 20 C This rDNase solution
6. Use sterile tips with filter Add 1 8 mercaptoethanol to Buffer RA1 Sample material Sample material not fresh Whenever possible use fresh material Reagents not applied or prepared properly Reagents not properly prepared Add the indicated volume of RNase free H O to the DNase vial and 96 100 ethanol to Buffer RA3 and Buffer RA4 Concentrate and mix Add 1 8 mercaptoethanol to Buffer RA1 Kit storage Poor RNA Store aliquots of the reconstituted DNase at 20 C quality or yield Store other kit components at room temperature Storage at low temperatures may cause salt precipitation Keep bottles tightly closed in order to prevent evaporation or contamination Sample material If using more than 10 cells use a shaker or a commercial homogenizer for optimal homogenization of the starting material MACHEREY NAGEL 04 2014 Rev 05 33 Total RNA isolation Problem Possible cause and suggestions Elution Be sure that all of the water gets into contact with the silica membrane No water drops should stick to the walls of the columns The membrane has to be wetted completely Poor RNA Clogging of the NucleoSpin RNA Binding Strips oor f yore quality or yield If using too much sample or if tissue lysate has not been successfully cleared clogging of the NucleoSpin RNA continued y an Binding Strips may appear Reduce sample amount and raise time for vacuum filtration or centrifugation steps to prevent
7. Buffer RA3 Wash Buffer RA4 and rDNase reaction mixture were prepared according to section 3 For each 50 uL 1 volume sample add 160 uL 3 2 volumes Buffer RA1 and 110 uL 2 2 volumes ethanol 96 100 to adjust conditions under which the RNA binds to the silica membrane It is possible to scale up the volumes The total volume of Buffer RA1 supplied in the kit is sufficient for a maximum of 300 uL Buffer RA1 per well See ordering information if additional buffer is required Note Do not exceed a total volume of 1 400 uL as this is the maximum capacity of the individual wells Mix by pipetting up and down at least 15 times and transfer samples to the wells of the NucleoSpin RNA Binding Plate Proceed with step 5 of the standard procedure Bind RNA to the silica membrane section 5 1 or 5 2 Note rDNase treatment may not be necessary depending on starting material and upstream application 32 MACHEREY NAGEL 04 2014 Rev 05 Total RNA isolation 6 Appendix 6 1 Troubleshooting Problem Possible cause and suggestions RNase contamination Create an RNase free environment on the worktable Clean trough reservoirs with appropriate solutions Wear gloves during all steps of the procedure Change gloves frequently Use of sterile disposable polypropylene tubes is recommended are D fill back d buffer f h h ir i degraded ma ie ack unused buffer from the trough reservoir into no RNA obtained
8. Last updated 07 2010 Rev 03 Please contact MACHEREY NAGEL GmbH amp Co KG Tel 49 0 24 21 969 270 e mail tech bio mn net com Trademarks BioRobot is a registered trademark of the Qiagen GmbH Corporation Geno Grinder is a registered trademark of SPEX SamplePrep Inc LightCycler is a registered trademark of a member of the Roche group NucleoSpin is a registered trademark of MACHEREY NAGEL GmbH amp Co KG SPEXCertiPrep is a registered trademark of SPEX SamplePrep Inc SYBR is a registered trademark of Molecular Probes Inc Vac Man is a registered trademark of Promega Corporation All used names and denotations can be brands trademarks or registered labels of their respective owner also if they are not special denotation To mention products and brands is only a kind of information i e it does not offend against trademarks and brands and can not be seen as a kind of recommendation or assessment Regarding these products or services we can not grant any guarantees regarding selection efficiency or operation 38 MACHEREY NAGEL 04 2014 Rev 05
9. Strips per 48 x 8 preps or 8x Round well Block with Cap Strips per 48 x 8 preps or 8x MN Square well Block Remarks Bind RNA to the membrane 8 x MN Wash Plate per 48 x 8 preps 2 x MN Square well Block MN Wash Plate minimizes the risk of cross contamination vacuum processing only For waste collection ji during centrifugation reusable MACHEREY NAGEL 04 2014 Rev 05 Total RNA isolation Protocol step Suitable consumables not supplied Remarks with the core kits Elute RNA 8x Rack of Tubes Strips with Cap Strips per 48 x 8 preps or 8x Round well Block with Cap Strips per 48 x 8 preps or 8x Elution Plate U bottom EEE For vacuum processing only or CE EF 8x Round well Block Low For processing under _ centrifugation 2 6 Automated processing on robotic platforms NucleoSpin 8 RNA can be used fully automated on many common laboratory workstations Please contact MN for the availability of scripts and general considerations about adapting NucleoSpin 8 RNA on a certain workstation Visit MN online at www mn net com or contact your local MACHEREY NAGEL distributor for technical support regarding hardware software setup instructions and selection of the protocol Several application notes of the NucleoSpin 8 RNA kit on various liquid handling instruments can also be found at www mn net com at Bioanalysis Literature
10. body is STRICTLY FORBIDDEN The respective user is liable for any and all damages resulting from such application DNA RNA PROTEIN purification products of MACHEREY NAGEL are suitable for IN VITRO USES ONLY ONLY MACHEREY NAGEL products specially labeled as IVD are also suitable for IN VITRO diagnostic use Please pay attention to the package of the product IN V TRO diagnostic products are expressiy marked as IVD on the packaging IF THERE IS NO IVD SIGN THE PRODUCT SHALL NOT BE SUITABLE FOR IN VITRO DIAGNOSTIC USE ALL OTHER PRODUCTS NOT LABELED AS IVD ARE NOT SUITED FOR ANY CLINICAL USE INCLUDING BUT NOT LIMITED TO DIAGNOSTIC THERAPEUTIC AND OR PROGNOSTIC USE No claim or representations is intended for its use to identify any specific organism or for clinical use included but not limited to diagnostic prognostic therapeutic or blood banking It is rather in the responsibility of the user or in any case of resale of the products in the responsibility of the reseller to inspect and assure the use of the DNA RNA protein purification products of MACHEREY NAGEL for a well defined and specific application MACHEREY NAGEL shall only be responsible for the product specifications and the performance range of MN products according to the specifications of in house quality control product documentation and marketing material This MACHEREY NAGEL product is shipped with documentation stating specifications and other technical
11. this If clogging happens during the run take the remaining lysate off the NucleoSpin RNA Binding Strips discard it and proceed with the desalting step Buffer RA3 The use of the NucleoSpin RNA Filter Strips is recommended to clear the lysate Contamination of RNA with genomic DNA rDNase not active e Reconstitute and store lyophilized rDNase according to instructions in section 3 Too much material used Reduce quantity of tissue Increase mixing cycles after adding Buffer RA4 to the lysate Suboptimal performance of RNA in downstream experiments Carry over of ethanol Be sure to remove all of ethanolic Buffer RA4 after the final washing step Dry the NucleoSpin RNA Binding Strips for at least 10 min with maximum vacuum or by 10 min centrifugation Insufficient Vacuum pressure is not sufficient vacuum Check if the vacuum manifold lid fits tightly on the manifold pressure base while vacuum is applied Buffer volumes are not enough Buffers are delivered in sufficient but limited amounts Ba Calculate the required buffer volumes and pour an additional Insufficient buffer volumes amount of 10 into the reservoirs if using a robotic platform Do not fill back unused buffer from reservoir into the bottle to avoid contaminations Ask technical service for extended buffer volumes 34 MACHEREY NAGEL 04 2014 Rev 05 Total RNA isolation Problem Possible cause and suggestions
12. type of sample up to 30 mg see also Table 2 can be processed Add 300 uL Buffer RA1 for tissue homogenization Follow the standard protocol for tissue samples If working with tissue or nuclease rich cells add 1 B mercaptoethanol to Buffer RA1 B mercaptoethanol supports the inhibition of RNases For sample homogenization and removal of cell debris it is recommended filtering the lysates through the NucleoSpin RNA Filter Strips not included in the kit before applying them to the NucleoSpin RNA Binding Strips Alternatively Centrifuge homogenized tissue samples for 5 min at maximum MACHEREY NAGEL 04 2014 Rev 05 11 Total RNA isolation g forces transfer supernatant to suitable plate and proceed with the standard protocol by adding Buffer RA4 Binding capacity of the membrane is up to 100 ug Depending on type of tissue and homogenization yield will differ and has to be tested individually Some typical results are depicted in the following table Table 3 Yields of total RNA with NucleoSpin 8 RNA Sample source Max starting material Max yield of total RNA Spleen 20 mg 50 ug Kidney 30 mg 45 ug Brain 30 mg 20 ug Liver 30 mg 80 ug Yield of total RNA depends strongly on the tissue and the effectiveness of lysis homogenization Therefore results may strongly vary Methods for sample homogenization Commercial homogenizers for example 2010 Geno Grinder COPS Diagnostics can be use
13. vacuum or water aspirator We recommend a vacuum of 0 2 to 0 4 bar reduction of atmospheric pressure The use of the NucleoVac Vacuum Regulator see ordering information is recommended Alternatively adjust the vacuum so that during the purification the sample flows through the column with a rate of 1 2 drops per second Depending on the amount of sample being used the vacuum times may need to be increased for complete filtration Additionally a suitable centrifuge for sample preparation steps may be required 8 MACHEREY NAGEL 04 2014 Rev 05 Total RNA isolation 2 5 Recommended accessories for use of the NucleoSpin 8 RNA Core Kit The NucleoSpin 8 RNA Core Kit provides buffers rDNase and NucleoSpin RNA Binding Strips Accessory plates e g lysis plates elution plates are not provided with the core kit The reduced kit composition along with a variety of separately available accessories allow optimal adjustment of the kit to individual user needs The user can select additional consumables according to his her requirements for highest flexibility For use of NucleoSpin 8 RNA Core Kit follow the standard protocols see section 5 1 and 5 2 Recommended accessories for use of the NucleoSpin 8 RNA Core Kit 48 x 8 preps are available from MACHEREY NAGEL see ordering information Protocol step Suitable consumables not supplied Lyse samples with the core kits 8x Rack of Tubes Strips with Cap
14. 0 mL 125 ml ng ae NucleoSpin RNA Binding 12 60 Strips blue rings Collection Tubes 1 5 ml 8 40 MN Wash Plates 2 5 including six Paper Sheets Racks of Tube Strips with Cap r 3 15 Strips MN Square well Blocks 2 2 Elution Plates U Bottom including one Self adhering 1 5 PE Foil User manual 1 1 1 For preparation of working solutions and storage conditions see section 3 For rDNase working solution during automated use 3 Is not used when following the centrifuge protocol in section 5 4 for the isolation of total RNA 4 Set consists of 1 Rack 12 Tube Strips with 8 tubes each and 12 Cap Strips 4 MACHEREY NAGEL 04 2014 Rev 05 Total RNA isolation 1 1 Kit contents continued NucleoSpin 8 RNA Core Kit 48 x 8 preps REF 740465 4 Lysis Buffer RA1 2x 125 mL Wash Buffer RA2 360 mL Wash Buffer RA3 Concentrate 2 x 100 mL Wash Buffer RA4 Concentrate 2x65 mL Reaction Buffer for rDNase 60 mL rDNase RNase free lyophilized 8 vials RNase free H O 2x 125 mL NucleoSpin RNA Binding Strips j 48 blue rings User manual 1 1 2 Reagents to be supplied by user 96 100 ethanol for preparation of working solutions see section 3 Reducing agent B mercaptoethanol or DTT dithiothreithol or TCEP BisTris Bis 2 hydroxyethyl imino tris hydroxymethyl methane as supplement for Lysis Buffer RA1 NucleoSpin RNA Filter Strips optional see section 2 7 For more detailed information regarding
15. 0 uL or 130 pL Buffer RA4 for lysates from cells grown in 96 well plates to each well of the lysis plate or tube Mix by pipetting up and down at least 10 15 times Note Buffer RA1 and Buffer RA4 have to be used in the same volume ratio Prepare NucleoVac 96 Vacuum Manifold Insert appropriate number of NucleoSpin RNA Binding Strips into a Column Holder A Close any unused openings of the Column Holder A with NucleoSpin Dummy Strips Note Make sure that the NucleoSpin RNA Binding Strips are inserted tightly into the Column Holder A Not properly inserted strips may prevent sealing when vacuum is applied to the manifold Insertspacers MTP MULTI 96 PLATE notched side up into the grooves located on the short sides of the manifold Insert the waste container into the center of the manifold Place the MN Wash Plate on top of the spacers in the manifold base Insert Column Holder A with inserted NucleoSpin RNA Binding Strips into the manifold lid and place lid on the manifold base 4 Transfer crude lysates to NucleoSpin RNA Binding Strips Apply the samples to the wells of the NucleoSpin RNA Binding Strips 5 Bind RNA to silica membrane Apply vacuum until all lysates have passed through the wells 0 2 bar 1 min Release the vacuum 6 Desalt silica membrane Desalt the membrane by adding 500 uL Buffer RA3 to each well and apply vacuum 0 2 bar 3 min until all buffer has passed through the wells Re
16. 3 5 600 6 000 x g 2 min 1 Cells grown in 96 well plates only MACHEREY NAGEL 04 2014 Rev 05 25 NucleoSpin 8 RNA centrifuge processing 7 Digest DNA 95 uL rDNase reaction mixture Room temperature 15 min 8 Wash silica membrane 500 pL RA2 5 600 6 000 x g 2 min 800 pL RA3 5 600 6 000 x g 2 min 500 pL RA4 5 600 6 000 x g 10 min 9 Dry NucleoSpin RNA Binding Plate Not necessary 10 Elute RNA 75 uL RNase free H O Incubate 2 min 5 600 6 000 x g 2 min 1 Prolonged centrifugation time is required to remove al traces of ethanol from last wash step 26 MACHEREY NAGEL 04 2014 Rev 05 NucleoSpin 8 RNA centrifuge processing Detailed protocol For hardware requirements refer to section 2 4 For use of the NucleoSpin 8 RNA Core Kit REF 740465 4 refer to section 2 5 regarding recommended accessories Before starting the preparation Check if Buffer RA3 Buffer RA4 and rDNase reaction mixture were prepared according to section 3 1 Harvest cells If cells have been grown in suspension aliquots of up to 1 x 10 cells can be transferred into the wells of a MN Square well Block included in the kit or into the wells of another suitable deep well plate or reaction tube e g Round well Block Tube Strips see ordering information Pellet cells by centrifugation 5 min 500 x g and remove the supernatant by pipetting 2 Lyse cells ot tis
17. a MN Square well Block included in the kit or another suitable deep well plate or reaction tube e g Round well Block Rack of Tube Strips see ordering information Pellet cells by centrifugation 5 min 500 x g and remove the supernatant by pipetting 2 Lyse cells or tissue Cells tissue samples Add 300 pL Buffer RA1 1 B mercaptoethanol vol vol to each sample Cells can be lysed by repeated pipetting up and down or vigorous shaking of the sealed closed plate or reaction tube For homogenization of tissue samples please refer to section 2 7 Note As alternative to B ME the reducing agent DTT or TCEP may be used Use a final concentration of 10 20 mM DTT or TCEP within the Lysis Buffer RA1 Optional It is recommended using the NucleoSpin RNA Filter Strips see ordering information for the clarification of tissue lysates Cells cultures grown in 96 well plates Discard culture medium and if necessary wash cells with PBS buffer once Add 130 uL Buffer RA1 1 B mercaptoethanol vol vol to the cells in each well of the cell culture plate Cells can be Iysed by repeated pipetting up and down or vigorous shaking of the sealed plate Note Use of B mercaptoethanol is recommended but not essential for most cell types also see section 2 7 MACHEREY NAGEL 04 2014 Rev 05 21 NucleoSpin 8 RNA vacuum processing 3 Prepare binding Depending on the volume of Buffer RA1 used in the lysis step add 30
18. acuum manifold 1 Reduction of atmospheric pressure MACHEREY NAGEL 04 2014 Rev 05 23 NucleoSpin 8 RNA vacuum processing Dry NucleoSpin RNA Binding Strips Remove any residual wash buffer from the NucleoSpin RNA Binding Strips If necessary tap the outlets of the NucleoSpin RNA Binding Strips onto a clean Paper Sheet Supplied with the MN Wash Plate or soft tissue until no further drops come out Insert the Column Holder A with inserted NucleoSpin RNA Binding Strips into the manifold lid and close the manifold Build up the vacuum with the valve closed Once the maximum vacuum 0 6 bar is achieved open the valve and apply vacuum for at least 10 min to dry the membrane completely This step is necessary to eliminate traces of ethanol Note The ethanol in Buffer RA4 inhibits enzymatic reactions and has to be removed completely before eluting DNA Finally release the vacuum 10 Elute RNA Place the Elution Plate U bottom onto the spacers MTP MULTI 96 PLATE of the vacuum manifold Pipette 75 uL RNase free H O directly to the bottom of each well 75 uL are recommended 50 130 uL are possible see section 2 8 Incubate for 2 min at room temperature Build up the vacuum with the valve closed Once the maximum vacuum 0 5 bar is achieved open the valve and apply vacuum for 1 min Alternatively elution into Tube Strips included in the kit or standard PCR plates is possible For
19. ate U bottom 740486 24 24 sets set consists of Elution Plate U bottom and Self adhering PE Foil Cap Strips 740478 48 740478 24 288 MN Wash Plate 740479 4 740479 24 24 Self adhering PE Foil 740676 50 NucleoVac 96 Vacuum Manifold 740681 1 NucleoVac Vacuum Regulator 740641 1 Starter Set A 740682 1 for processing NucleoSpin 8 well strips on NucleoVac 96 Vacuum Manifold Starter Set C 740684 1 for processing NucleoSpin 8 well strips under centrifugation MN Frame 740680 1 Visit www mn net com for more detailed product information 6 3 Product use restriction warranty NucleoSpin 8 RNA Core Kit components are intended developed designed and sold FOR RESEARCH PURPOSES ONLY except however any other function of the product being expressly described in original MACHEREY NAGEL product leaflets MACHEREY NAGEL products are intended for GENERAL LABORATORY USE ONLY MACHEREY NAGEL products are suited for QUALIFIED PERSONNEL ONLY MACHEREY NAGEL products shall in any event only be used wearing adequate PROTECTIVE CLOTHING For detailed information please refer to the respective Material Safety Data Sheet of the product MACHEREY NAGEL products shall 36 MACHEREY NAGEL 04 2014 Rev 05 Total RNA isolation exclusively be used in an ADEQUATE TEST ENVIRONMENT MACHEREY NAGEL does not assume any responsibility for damages due to improper application of our products in other fields of application Application on the human
20. d for sample homogenization Use of NucleoSpin RNA Filter Strips For sample homogenization and to prevent clogging of the NucleoSpin RNA Binding Strips the use of the NucleoSpin RNA Filter Strips is recommended under vacuum or centrifugation Centrifugation Insert desired number of NucleoSpin RNA Filter Strips into the Column Holder C see ordering information and place it on a MN Square well Block Starter Set C see ordering information Transfer lysates in Buffer RA1 to the wells of the NucleoSpin RNA Filter Strips Centrifuge at 5 600 6 000 x g until all samples have passed the filter approx 5 min Start the RNA purification procedure with the filtrate collected in the MN Square well Block Vacuum Insert spacers SQUARE WELL BLOCK into the NucleoVac 96 Vacuum Manifold Put a Square well Block in the manifold and close the manifold with the manifold lid Insert desired number of NucleoSpin RNA Filter Strips into the Column Holder A Starter Set A see ordering information and place it on top of the manifold 12 MACHEREY NAGEL 04 2014 Rev 05 Total RNA isolation Transfer the sample Iysate in Buffer RA1 to the filter and apply vacuum until the lysates have passed the filter Start the RNA purification procedure with the flow through collected in the MN Square well Block Please note that the dead volume of the NucleoSpin RNA Filter Strips is rather large compared to the processing under centri
21. ding Strips All other steps may be processed with needles Adjust vacuum times and strength if necessary Take care that the volume of the rDNase reaction mixture is pipetted to the middle of the well 30 MACHEREY NAGEL 04 2014 Rev 05 Automated purification using laboratory automation workstations For increased RNA concentration dispense at least 75 uL of RNase free H O to the membrane Lower volumes of elution buffer will cause innomogeneous results By using higher volumes of dispensed water the concentration of eluted RNA will decrease but the efficiency of elution will increase Alternatively the elution can be performed in a centrifuge to reduce the volume of water needed for elution thus increasing the concentration of the RNA Stop the protocol after the vacuum drying step Remove the NucleoSpin RNA Binding Strips and tap it on a sheet of filter paper to remove residual wash buffer Place the NucleoSpin RNA Binding Strips on top of a Rack of Tube Strips MACHEREY NAGEL 04 2014 Rev 05 31 Clean up of total RNA 5 4 Clean up of total RNA This support protocol is designed for clean up of pre purified RNA samples e g from extractions using phenol chloroform based purification procedures precipitation protocols or following enzymatic reactions The NucleoSpin 8 RNA clean up procedure will eliminate traces of organic solvents salts or enzymes Before starting the preparation Check if Wash
22. ell small scale purification of total RNA from tissue or cells Fresh frozen or stabilized sample material can be processed The NucleoSpin 8 RNA kits can be used manually under vacuum or under centrifugation The NucleoSpin 8 RNA kits can be used fully automated on common laboratory workstations see section 2 6 The kits provide reagents and consumables for purification of up to 100 ug highly pure total RNA suitable for direct use in standard molecular biology applications like RT PCR TaqMan Northern Blot or microarray analysis The NucleoSpin 8 RNA kits yield RNA of highest purity and integrity Using the NucleoSpin 8 RNA kits allows for simultaneous processing of up to 48 samples typically within less than 45 minutes Actual automated processing time depends on the configuration of workstation used 2 3 Yield and quality of total RNA NucleoSpin 8 RNA can be used under vacuum or in a centrifuge In a centrifuge however slightly higher yields are possible because of the higher amount of starting material that can be processed and the reduced dead volume of the membrane 6 MACHEREY NAGEL 04 2014 Rev 05 Total RNA isolation Typical amounts of starting material and anticipated yields are shown in Table 2 Please note that yield of total RNA strongly depends on the starting material and on complete lysis homogenization Results may vary For more information about the lysis homogenization proces
23. elution in Tube Strips place the Rack of Tube Strips on the spacers MICROTUBE STRIPS inside the manifold Elution into PCR plates can be performed by placing a PCR plate onto a MN Square well Block placed on the spacers SQUARE WELL BLOCK in the manifold 1 Reduction of atmospheric pressure 24 MACHEREY NAGEL 04 2014 Rev 05 NucleoSpin 8 RNA centrifuge processing 5 2 NucleoSpin 8 RNA centrifuge processing For hardware requirements refer to section 2 4 For detailed information on each step see page 30 For use of the NucleoSpin 8 RNA Core Kit REF 740465 4 refer to section 2 5 regarding recommended accessories Before starting the preparation Check if Buffer RA3 Buffer RA4 and rDNase reaction mixture were prepared according to section 3 Protocol at a glance 1 Harvest cells 500 x g 5 min by celgorussue 300 pL RA1 cells tissue Optional If using tissue samples or 3 pL B ME large number of cells clearing of lysate r with the NucleoSpin RNA Filter Strips is recommended 130 uL RA1 cells Centrifuge 5 600 6 00 x g for 5 min 1 3 uL EME 3 Prepare binding 300 uL RA4 cells tissue Mix by pipetting up and down at least or ln 130 uL RA4 cells 4 Transfer crude lysates to NucleoSpin RNA Binding Strips 5 Bind RNA to silica membrane of the 5 600 6 000 x g NucleoSpin RNA Binding Strips 2 min 6 Desalt silica membrane by washing 500 pL RA
24. er disruption samples can be stored at 70 C in Lysis Buffer RA1 Frozen samples are stable for up to 6 months Frozen samples in Buffer RA1 should be thawed completely and centrifuged before starting with the isolation of total RNA If larger cell numbers or large amounts of tissue are used a filtration step of the RA1 lysate through the NucleoSpin RNA Filter Strips see ordering information is recommended for optimal homogenization and removal of particles Cell culture Up to 2x 10 cells can be processed under vacuum Using a centrifuge up to 1x 107 cells can be processed Transfer the cell suspension to a suitable square well block not included in the kit and centrifuge for 5 min at 500 x g The supernatant has to be removed completely Lyse cells by addition of 300 uL Buffer RA1 Follow the standard protocol for cell cultures For adherent cell cultures in 96 well format make sure that the culture medium is completely removed Lyse cells by addition of 130 uL Buffer RA1 Follow the standard protocol for cell cultures grown in 96 well plates If using more than 1x 10 cells it is recommended using a commercial homogenizer for lysis with Buffer RA1 in order to reduce viscosity To prevent the NucleoSpin RNA Binding Strips from clogging it is also recommended filtrating the lysates through the NucleoSpin RNA Filter Strips not included in the kit before applying them to the NucleoSpin RNA Binding Strips Depending on the
25. fication using common laboratory automation workstations Before starting the preparation e Check if Wash Buffer RA3 Wash Buffer RA4 and rDNase reaction mixture were prepared according to section 3 Note For ready to run robot scripts and general information about automation please contact your local distributor or MN directly 1 Place the plastic equipment like plates and the assembled vacuum manifold at the locations of the robotic platforms as specified in the individual robotic programs 2 Add sufficient buffer to the reservoirs or place the buffer bottles at the corresponding positions on the robot worktable Calculate the required buffer volumes and pour an additional amount of 10 into the reservoirs Buffers are delivered in sufficient but limited amounts and should not be wasted Do not fill back unused buffer into the bottle 3 Harvest cells If cells have been grown in suspension aliquots of up to 2 x 10 cells can be transferred into the wells of a deep well plate Pellet cells by centrifugation for 5 min at 500 x 9 Tissue samples For harvesting and homogenization of tissue samples please refer to section 2 7 4 Place the samples at the appropriate position of the robot worktable 5 Select method for total RNA purification and start the run Seal unused wells with Self adhering PE Foil see ordering information Use disposable tips with filter for the transfer of sample to the NucleoSpin RNA Bin
26. fugation thus processing under vacuum is only recommended when complete automation is desired 2 8 Elution of pure total RNA Due to dead volume of the silica membrane by using vacuum please notice that the difference between the dispensed elution buffer and the recovered elution buffer containing total RNA is approx 20 uL For RNA elution a volume of 50 130 uL nuclease free water is recommended Higher RNA concentrations are obtained when using a dispense volume of 50 uL however higher elution efficiencies are obtained when using dispense volumes of gt 100 UL Recovered elution volume Dispensed elution volume 20 uL Elution is possible under vacuum and in a centrifuge without any cross contamination see Figure 1 To achieve this vacuum settings during the elution have to be adjusted carefully smooth elution to avoid splattering of liquid 0 04 A u A 2 0 00 8 well with sample o well without sample E an a een ee pi HE ar aa 30 B Cycle number a rak aN a Kan kani van gan dF Figure 1 HeLa cells 5 x 10 each were pelleted in a culture plate in a chess board pattern Total RNA was prepared using NucleoSpin 96 RNA a RT PCR detection of total RNA was performed with 1 5 uL of the eluate total eluate 80 uL in a LightCycler 0 5 uM GAPDH primer LightCycler RNA Amplification Kit Hybridization Probes b 20 uL of the LightCycler assay were loaded on a 2 agarose gel
27. information MACHEREY NAGEL warrants to meet the stated specifications MACHEREY NAGEL s sole obligation and the customer s sole remedy is limited to replacement of products free of charge in the event products fail to perform as warranted Supplementary reference is made to the general business terms and conditions of MACHEREY NAGEL which are printed on the price list Please contact us if you wish to get an extra copy There is no warranty for and MACHEREY NAGEL is not liable for damages or defects arising in shipping and handling transport insurance for customers excluded or out of accident or improper or abnormal use of this product defects in products or components not manufactured by MACHEREY NAGEL or damages resulting from such non MACHEREY NAGEL components or products MACHEREY NAGEL makes no other warranty of any kind whatsoever and SPECIFICALLY DISCLAIMS AND EXCLUDES ALL OTHER WARRANTIES OF ANY KIND OR NATURE WHATSOEVER DIRECTLY OR INDIRECTLY EXPRESS OR IMPLIED INCLUDING WITHOUT LIMITATION AS TO THE SUITABILITY REPRODUCTIVITY DURABILITY FITNESS FOR A PARTICULAR PURPOSE OR USE MERCHANTABILITY CONDITION OR ANY OTHER MATTER WITH RESPECT TO MACHEREY NAGEL PRODUCTS MACHEREY NAGEL 04 2014 Rev 05 37 Total RNA isolation In no event shall MACHEREY NAGEL be liable for claims for any other damages whether direct indirect incidental compensatory foreseeable consequential or special including but not limited t
28. is stable for at least six months Do not freeze thaw the aliquots more than three times Prepare rDNase reaction mixture For each sample to be processed mix 10 uL reconstituted rDNase with 90 uL Reaction Buffer for rDNase Wash Buffer RA3 Add the indicated volume of 96 100 ethanol to the Buffer RA3 Concentrate Indicate that ethanol is added by marking the bottle label Store Wash Buffer RA3 at room temperature 18 25 C for at least one year Wash Buffer RA4 Add the indicated volume of 96 100 ethanol to the Buffer RA4 Concentrate Mark the bottle label to indicate that ethanol has been added Store Wash Buffer RA4 at room temperature 18 25 C for at least one year 14 MACHEREY NAGEL 04 2014 Rev 05 Total RNA isolation NucleoSpin 8 RNA 12 x 8 preps 60 x 8 preps REF 740698 740698 5 Wash Buffer RA3 1x 50 mL 2x 100 mL Concentrate Add 200 mL ethanol Add 400 mL ethanol to each bottle Wash Buffer RA4 1x65 mL 2x65 mL Concentrate Add 150 mL ethanol Add 150 mL ethanol to each bottle NucleoSpin 8 RNA Core Kit 48 x 8 preps REF 740465 4 Wash Buffer RA3 2x 100 mL Concentrate Add 400 mL ethanol to each bottle Wash Buffer RA4 2x65 mL Concentrate Add 150 mL ethanol to each bottle MACHEREY NAGEL 04 2014 Rev 05 15 Total RNA isolation 4 Safety instructions The following components of the NucleoSpin 8 RNA and NucleoSpin 8 RNA Core kits contain hazardous contents Wear glove
29. lease the vacuum 1 Reduction of atmospheric pressure 22 MACHEREY NAGEL 04 2014 Rev 05 NucleoSpin 8 RNA vacuum processing Digest DNA Prepare rDNase reaction mixture as described in section 3 Pipette 95 uL rDNase reaction mixture directly to the bottom of each well of the NucleoSpin RNA Binding Strips Do not touch the silica membrane with the pipette tips Incubate at room temperature for 15 min Be sure that all of the rDNase reaction mixture gets into contact with the silica membrane and that the membrane is completely wetted Wash silica membrane Add 500 pL Buffer RA2 to each well of the NucleoSpin RNA Binding Strips Apply vacuum 0 2 bar 1 min until all buffer has passed through the wells Release the vacuum Add 800 uL Buffer RA3 to each well of the NucleoSpin RNA Binding Strips Apply vacuum 0 2 bar 1 min until all buffer has passed through the wells Release the vacuum Add 500 pL Buffer RA4 to each well of the NucleoSpin RNA Binding Strips Apply vacuum 0 2 bar 1 min until all buffer has passed through the wells Release the vacuum Remove MN Wash Plate After the final wash step close the valve release the vacuum and remove the Column Holder A with inserted NucleoSpin RNA Binding Strips from the vacuum manifold Put it on a clean paper towel to remove residual EtOH containing wash buffer Remove manifold lid MN Wash Plate and waste container from the v
30. m manifold Binding Washing steps Step 4 Place the NucleoSpin Binding Strips inserted the Column Holder A on top of the manifold lid Unused rows have to be filled with NucleoSpin Dummy Strips Step 3 Place the manifold lid on top of the manifold base Step 2 Place the MN Wash Plate in the manifold Step 1 Insert spacers MTP MULTI 96 PLATE and waste container in the manifold base Final setup Step 4 Place the NucleoSpin Binding Strips inserted the Column Holder A on top of the manifold lid Unused rows have to be filled with NucleoSpin Dummy Strips Step 3 Place the manifold lid on top of the manifold base Step 2 Place the Elution Plate in the manifold Step 1 Insert spacers MTP MULTI 96 PLATE in the manifold base Final setup 20 MACHEREY NAGEL 04 2014 Rev 05 NucleoSpin 8 RNA vacuum processing Detailed protocol For hardware requirements refer to section 2 4 For detailed information regarding the vacuum manifold setup see page 23 For use of the NucleoSpin 8 RNA Core Kit REF 740465 4 refer to section 2 5 regarding recommended accessories Before starting the preparation e Check if Buffer RA3 Buffer RA4 and rDNase reaction mixture were prepared according to section 3 1 Harvest cells If cells have been grown in suspension aliquots of up to 2 x 10 cells can be transferred into wells of
31. nal If using tissue samples or large number of cells clearing of lysate with the NucleoSpin RNA Filter Strips is recommended Transfer cleared lysate to MN Square well Block 300 uL RA1 cells tissue 3 pL B ME or 130 uL RA1 cells 1 3 pL B ME 3 Prepare binding Mix by pipetting up and down at least 10 15 times 300 uL RA4 cells tissue or 130 uL RA4 cells Prepare vacuum manifold 4 Transfer crude lysates to NucleoSpin RNA Binding Strips 5 Bind RNA to silica membrane of the NucleoSpin RNA Binding Strips 0 2 bar 1 min 1 Cells grown in 96 well plates only Reduction of atmospheric pressure 18 MACHEREY NAGEL 04 2014 Rev 05 NucleoSpin 8 RNA vacuum processing 6 Desalt silica membrane by washing 500 pL RA3 0 2 bar 3 min 7 Digest DNA 95 pL rDNase reaction mixture Room temperature 15 min 8 Wash silica membrane 500 pL RA2 800 pL RA3 500 uL RA4 0 2 bar 1 min each step Remove MN Wash Plate 9 Dry NucleoSpin RNA Binding Strips Maximum vacuum 0 6 bar by applying vacuum 10 min Optional Dry the outlets of the NucleoSpin RNA Binding Strips by placing it on a Paper Sheet before applying vacuum 10 Elute RNA 75 uL RNase free H O Incubate 2 min 0 5 bar 1 min 1 Reduction of atmospheric pressure MACHEREY NAGEL 04 2014 Rev 05 19 NucleoSpin 8 RNA vacuum processing Setup of vacuu
32. o loss of use revenue or profit whether based upon warranty contract tort including negligence or strict liability arising in connection with the sale or the failure of MACHEREY NAGEL products to perform in accordance with the stated specifications This warranty is exclusive and MACHEREY NAGEL makes no other warranty expressed or implied The warranty provided herein and the data specifications and descriptions of this MACHEREY NAGEL product appearing in MACHEREY NAGEL published catalogues and product literature are MACHEREY NAGEL s sole representations concerning the product and warranty No other statements or representations written or oral by MACHEREY NAGEL s employees agent or representatives except written statements signed by a duly authorized officer of MACHEREY NAGEL are authorized they should not be relied upon by the customer and are not a part of the contract of sale or of this warranty Product claims are subject to change Therefore please contact our Technical Service Team for the most up to date information on MACHEREY NAGEL products You may also contact your local distributor for general scientific information Applications mentioned in MACHEREY NAGEL literature are provided for informational purposes only MACHEREY NAGEL does not warrant that all applications have been tested in MACHEREY NAGEL laboratories using MACHEREY NAGEL products MACHEREY NAGEL does not warrant the correctness of any of those applications
33. on containing Column Holders C NucleoSpin Dummy Strips MN Square well Blocks and Rack of Tube Strips is required For centrifugation with Column Holder C with inserted NucleoSpin RNA Binding Strips stacked on a MN Square well Block or Rack of Tube Strips a microtiter plate centrifuge is required which is able to accommodate the above stacked plates and reach accelerations of 5 600 6 000 x g bucket height 85 mm Regarding waste collection suitable consumables e g MN Square well Blocks are necessary and they are not included in the kit For the most convenient handling without the need of emptying and reusing the MN Square well Blocks we recommend using six MN Square well Blocks if two column holders are processed at once see ordering information Alternatively it is possible to empty the MN Square well Blocks after every centrifugation step reducing the amount of MN Square well Blocks needed Vacuum processing For processing 8 well strips under vacuum the Starter Set A see ordering infomation containing Column Holders A and NucleoSpin Dummy Strips is required For automation on laboratory platforms with standard 96 well plate manifolds the use of Starter Set Ais also required The NucleoSpin 8 RNA kit can be used manually with the NucleoVac 96 Vacuum Manifold see ordering information Establish a reliable vacuum source for the NucleoVac 96 Vacuum Manifold The manifold may be used with a vacuum pump house
34. rates toxic gas Entwickelt bei Ber hrung mit S ure giftige Gase Hazard labeling not neccessary if quantity per bottle below 125g or ml certificate of exemption according to 67 548 EEC Art 25 1999 45 EC Art 12 and German GefStoffV 20 3 and TRGS 200 7 1 For further information see Material Safety Data Sheet 16 MACHEREY NAGEL 04 2014 Rev 05 Total RNA isolation Precaution phrases P 210 P 233 P 260 P 261 P 273 P 280 P 301 312 P 302 352 P 304 340 P 330 P 333 313 P 342 311 P 403 235 P 363 Keep away from heat hot surfaces sparks open flames and other ignition sources No smoking Von Hitze hei8en Oberfl chen Funken offenen Flammen sowie anderen Z ndquellenarten fernhalten Nicht rauchen Keep container tightly closed Beh lter dicht verschlossen halten Do not breathe vapours Dampf nicht einatmen Avoid breathing dust Einatmen von Staub vermeiden Avoid release to the environment Freisetzung in die Umwelt vermeiden Wear protective gloves eye protection Schutzhandschuhe Augenschutz tragen IF SWALLOWED Call a POISON CENTER doctor if you feel unwell BEI VERSCHLUCKEN Bei Unwohlsein GIFTINFORMATIONSZENTRUM Arzt anrufen IF ON SKIN Wash with plenty of water BEI KONTAKT MIT DER HAUT Mit viel Wasser waschen IF INHALED Remove victim to fresh air and keep at rest in a position comfort able for breathing Bei Einatmen An die f
35. rische Luft bringen und in einer Position ruhigstellen die das Atmen erleichtert Rinse mouth Mund aussp len IF skin irritation or a rash occurs Get medical advice attention Bei Hautreizung oder ausschlag Arztlichen Rat einholen rztliche Hilfe hinzuziehen If experiencing respiratory symptoms Call a POISON CENTER doctor Bei Symptomen der Atemwege GIFTINFORMATIONSZENTRUM Arzt anrufen Store in a well ventilated place Keep cool K hl an einem gut bel fteten Ort augbewahren Wash contaminated clothing before reuse Kontaminierte Kleidung vor erneutem Tragen waschen For further information please see Material Safety Data Sheets www mn net com Weiterf hrende Informationen finden Sie in den Sicherheitsdatenbl ttern www mn net com MACHEREY NAGEL 04 2014 Rev 05 17 NucleoSpin 8 RNA vacuum processing 5 Protocols 5 1 NucleoSpin 8 RNA vacuum processing For hardware requirements refer to section 2 4 For detailed information regarding the vacuum manifold setup see page 23 For detailed information about each step see page 24 For use of the NucleoSpin 8 RNA Core Kit REF 740465 4 refer to section 2 5 regarding recommended accessories Before starting the preparation Check if Buffer RA3 Buffer RA4 and rDNase reaction mixture were prepared according to section 3 Protocol at a glance 1 Harvest cells 500 x g 5 min 2 Lyse cells or tissue Optio
36. rude lysates to NucleoSpin RNA Binding Strips Insert desired number of NucleoSpin RNA Binding Strips into the Column Holder C and place on it a MN Square well Block for collection of flow through If using more than one block label the column holders for later identification Transfer lysates to the wells of the NucleoSpin RNA Binding Strips 5 Bind RNA to silica membrane Centrifuge for 2 min at 5 600 6 000 x g 6 Desalt silica membrane Desalt the membrane by adding 500 uL Buffer RA3 to each well and centrifuge for 2 min at 5 600 6 000 x g Empty MN Square well Block 7 Digest DNA Prepare rDNase reaction mixture as described in section 3 Place Column Holder C with the NucleoSpin RNA Binding Strips on the MN Square well Block Pipette 95 wl rDNase reaction mixture directly to the bottom of each well of the NucleoSpin RNA Binding Strips Do not touch the silica membrane with the pipette tips Incubate at room temperature for 15 min Be sure that all of the rDNase reaction mixture gets into contact with the silica membrane and that the membrane is completely wetted 8 Wash silica membrane Add 500 ul Buffer RA2 to each well of the NucleoSpin RNA Binding Strips Place the Column Holder C with the NucleoSpin RNA Binding Strips on the MN Square well Block into the rotor bucket and centrifuge for 2 min at 5 600 6 000 x g Empty MN Square well Block 28 MACHEREY NAGEL 04 2014 Rev 05 NucleoSpin 8 RNA centrifuge processing
37. s see section 2 7 Table 2 Kit specifications at a glance Parameter NucleoSpin 8 RNA Technology Silica membrane technology Format 8 well strips Processing Manual or automated vacuum or centrifugation Animal tissue Cell culture Vacuum Centrifuge Vacuum Centrifuge Max sample size 10 30 mg 30 mg 2x 10 cells 1x 10 cells Typical yield Up to 40 ug Upto 100 ug Upto 20 ug Up to 100 ug Fragment size gt 200 nt Ago Asso 1 9 2 1 Typical RIN RNA Sharp rRNA bands with no substantial degradative integrity number bands visibile 28S 18S 2 1 Excellent RNA Integrity Number RIN values typically gt 9 cells 7 tissue Typical concentration 50 200 ng uL Elution volume 50 130 uL Preparation time 45 min 6 strips Binding capacity 100 ug The final concentration of eluted RNA is 50 500 ng uL depending on elution volume and starting material Suitable elution volumes range from 50 uL to 130 uL For RNA purity typically the Azgo Azgo ratio is 1 9 2 1 Isolated RNA is of highest quality and integrity indicating a highly efficient inactivation of RNases and gentle purification MACHEREY NAGEL 04 2014 Rev 05 7 Total RNA isolation 2 4 Required hardware NucleoSpin 8 RNA can be processed under vacuum or centrifugation Certain hardware for processing is required Centrifugation For processing the 8 well strips under centrifugation the Starter Set C see ordering informati
38. s and goggles and follow the safety instructions given in this section GHS classification Only harmful features do not need to be labeled with H and P phrases up to 125 mL or 125 g Mindergef hrliche Eigenschaften m ssen bis 125 mL oder 125 g nicht mit H und P S tzen gekennzeichnet werden Component Hazard contents GHS symbol Hazard Precaution phrases phrases Inhalt Gefahrstoff GHS Symbol H S tze P S tze RA1 Guanidinium thiocyanate Warning 302 412 260 273 30 60 EUH031 301 312 330 Guanidiniumthiocyanat Achtung 30 60 RA2 Guanidinium thiocyanate Warning 226 302 210 233 260 30 60 ethanol 20 412 273 301 312 35 EUH031 330 403 235 Guanidiniumthiocyanat Achtung 30 60 Ethanol 20 35 rDNase rDNase Iyophilized Danger 317 334 261 280 RNase free rDNase lyophilisiert lt Gefahr 302 352 304 340 333 313 342 311 363 Hazard phrases H 226 Flammable liquid and vapour Fl ssigkeit und Dampf entz ndbar H 302 Harmful if swallowed Gesundheitssch dlich bei Verschlucken H 317 May cause an allergic skin reaction Kann allergische Hautreaktionen verursachen H 334 May cause allergy or asthma symptoms or breathing difficulties if inhaled Kann bei Einatmen Allergie asthmaartige Symptome oder Atembeschwerden verursachen H 412 Harmful to aquatic life with long lasting effects Sch dlich f r Wasserorganismen mit langfristiger Wirkung EUH031 Contact with acids libe
39. special hardware required for centrifuge or vacuum processing please see section 2 4 For ordering information please see section 6 2 For recommended accessories for use of the flexible NucleoSpin 8 RNA Core Kit reduced kit composition REF 740465 4 please see section 2 5 1 For preparation of working solutions and storage conditions see section 3 MACHEREY NAGEL 04 2014 Rev 05 5 Total RNA isolation 2 Product description 2 1 The basic principle One of the most important aspects while working with RNAis to prevent RNA degradation during the isolation procedure With the NucleoSpin 8 RNA kits cells or tissues are lysed by incubation in a solution containing large amounts of chaotropic salt This lysis buffer immediately inactivates RNases which are present in virtually all biological materials In combination with Buffer RA4 it furthermore creates the appropriate binding conditions that favor RNA adsorption to the silica membrane Contaminating DNA which is also bound to the silica membrane is removed by directly applying DNase onto the silica membrane during the preparation RNase free recombinant DNase is supplied with the kit Salts proteins and other cellular components are removed by simple washing steps with three different buffers Finally pure RNA is eluted under low ionic strength conditions with RNase free water supplied 2 2 Kit specifications The NucleoSpin 8 RNA kits are designed for fast 8 w
40. sue Cells tissue samples Add 300 uL Buffer RA1 1 mercaptoethanol vol vol to each sample Cells can be lysed by pipetting up and down repeatedly or vigorous shaking of the sealed closed plate or reaction tube For homogenization of tissue samples please refer to section 2 7 Note As alternative to B ME the reducing agent DTT or TCEP may be used Use a final concentration of 10 20 mM DTT or TCEP within the Lysis Buffer RA1 Optional It is recommended using the NucleoSpin RNA Filter Strips see ordering information for the clarification of tissue lysates Cells cultures grown in 96 well plates Discard culture medium and if necessary wash cells with PBS buffer once Add 130 uL Buffer RA1 1 B mercaptoethanol vol vol to the cells in each well of the cell culture plate Cells can be lysed by pipetting up and down repeatedly or vigorous shaking of the sealed plate Note Use of B mercaptoethanol is recommended but not essential for most cell types also see section 2 7 MACHEREY NAGEL 04 2014 Rev 05 27 NucleoSpin 8 RNA centrifuge processing 3 Prepare binding Depending on the volume of Buffer RA1 used in the lysis step add 300 pL Buffer RA4 or 130 uL Buffer RA4 for lysates from cells grown in 96 well plates to each well of the lysis plate or tube Mix by pipetting up and down at least 10 15 times Note Buffer RA1 and Buffer RA4 have to be used in the same volume ratio 4 Transfer c
Download Pdf Manuals
Related Search