RNA from blood - MACHEREY
Contents
1. Problem Possible cause and suggestions RNase contamination Create an RNase free environment on the worktable Clean through reservoirs with appropriate solutions Wear gloves during all steps of the procedure Change gloves frequently Use of sterile disposable polypropylene tubes is recommended RNA is degraded no Do not fill back unused buffer from the trough reservoir into RNA obtained the bottle Use sterile tips with filter Sample material Sample material was not fresh Whenever possible use fresh blood samples Reagents not applied or prepared properly Reagents were not properly prepared Add the indicated volume of RNase free H O to the rDNase vial and 96 100 ethanol to Buffer RB3 and Buffer RB4 Concentrate and mix see section 3 Kit storage Store aliquots of the reconstituted rDNase at 20 C Store other kit components at room temperature Storage at low Poor RNA temperatures may cause salt precipitation quality or yield Keep bottles tightly closed in order to prevent evaporation or contamination Elution e Be sure that all of the water comes into contact with the silica membrane There should not be any water droplets on the walls of the columns The membrane needs to be completely wet Elute two times e g 2 x 50 uL MACHEREY NAGEL 05 2014 Rev 02 23 RNA from blood Problem Possible cause and suggestions Clogged wells Insufficient vacuum Prolong vacuum time to2. Add 800 pL Buffer RB3 to each well of the NucleoSpin RNA Blood Binding Plate Apply vacuum 0 2 bar 1 min until all buffer has passed through the wells Release the vacuum Reduction of atmospheric pressure 16 MACHEREY NAGEL 05 2014 Rev 02 NucleoSpin 96 RNA Blood vacuum processing Add 500 uL Buffer RB4 to each well of the NucleoSpin RNA Blood Binding Plate 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 NucleoSpin RNA Blood Binding Plate from the vacuum manifold Put it on a clean paper towel to remove residual ethanol containing wash buffer Remove manifold lid MN Wash Plate and waste container from the vacuum manifold Dry silica membrane Remove any residual wash buffer from the NucleoSpin RNA Blood Binding Plate If necessary tap the outlets of the NucleoSpin RNA Blood Binding Plate onto a clean Paper Sheet supplied with the MN Wash Plate or soft tissue until there are no more drops observed Insert the NucleoSpin RNA Blood Binding Plate 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
3. RNA from blood User manual NucleoSpin 96 RNA Blood May 2014 Rev 02 MACHEREY NAGEL www mn net com RNA from blood Table of contents 1 Components 1 1 Kit contents 1 2 Reagents to be supplied by user 4 2 Product description 5 2 1 The basic principle 5 2 2 Kit specifications 5 2 3 Required hardware 6 2 4 Automated processing on robotic platforms 7 2 5 Handling preparation and storage of starting material 7 3 Storage conditions and preparation of working solutions 8 4 Safety instructions 10 5 Protocols 12 5 1 NucleoSpin 96 RNA Blood vacuum processing 12 5 2 NucleoSpin 96 RNA Blood centrifuge processing 18 6 Appendix 23 6 1 Troubleshooting 23 6 2 Ordering information 26 6 3 Product use restriction warranty 27 MACHEREY NAGEL 05 2014 Rev 02 3 RNA from blood 1 Components 1 1 Kit contents NucleoSpin 96 RNA Blood 2 x 96 preps 4 x 96 preps REF 740225 2 740225 4 Lysis Buffer DL 100 mL 2x 100 mL Wash Buffer RB2 160 mL 360 mL Wash Buffer RB3 Concentrate 100 mL 2x 100 mL Wash Buffer RB4 Concentrate 65 mL 2x 65 mL RNase free H O 125 mL 125 mL Reaction Buffer for rDNase 60 mL 2 x 60 mL rDNase RNase free lyophilized Liquid Proteinase K NucleoSpin RNA Blood Binding Plates blue rings MN Wash Plates Square well Blocks including one self adhering PE Foil Elution Plates U bottom including one Self adhering PE Foil Round well Block Low including one Self
4. 5 10 min at 0 4 to 0 6 bar reduction of atmospheric pressure step 4 Bind RNA to silica membrane Prolong centrifugation step to 5 min at 5 600 6 000 x g Colored membrane after last wash step with RB3 Insufficient washing Repeat RB3 wash step 800 uL Contamination rDNase not active Reconstitute and store lyophilized rDNase according to the instructions in section 3 Too much material used of RNA with Reduce quantity of blood used genomic DNA Increase mixing cycles after addition of Buffer RB4 to the lysate Do not release vacuum until all buffer has passed through important after every step Carry over of ethanol Suboptimal e Be sure to remove all ethanolic Buffer RB4 after the final performance washing step prior elution Dry the NucleoSpin RNA Blood of RNA in Binding Plates for at least 10 min with maximum vacuum or downstream by 10 min centrifugation experiments Do not release vacuum until all buffer has passed through important after every step Vacuum pressure is not sufficient Check if the vacuum manifold lid fits tightly on the manifold base if vacuum is turned on Close unused rows with Self adhering PE Foil supplied 24 MACHEREY NAGEL 05 2014 Rev 02 RNA from blood Problem Possible cause and suggestions Buffer volumes are not sufficient Cross contamination e Buffers are delivered in sufficient but limited amounts Calculate the
5. 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 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
6. adhering PE Foil User manual 4 vials size D 2x 1 25 mL 2 1 2 Reagents to be supplied by user 96 100 ethanol 1 Patent pending 2 For preparation of working solutions and storage conditions see section 3 8 vials size D 4x 1 25 mL 4 3 Includes six paper sheets They are not used when following the centrifuge protocol in section 5 2 for the isolation of total RNA 4 MACHEREY NAGEL 05 2014 Rev 02 RNA from blood 2 Product description 2 1 The basic principle The NucleoSpin 96 RNA Blood kit offers a direct total blood lysis from up to 400 uL whole blood collected in standard e g EDTA Na citrate or Li heparin blood collection tubes One of the most important aspects in RNA isolation is to prevent RNA degradation during the isolation procedure With the NucleoSpin 96 RNA Blood method leukocytes the main source of RNA in whole blood and other blood cells are lysed by incubating the whole blood in a solution containing large amounts of chaotropic ions This lysis buffer immediately inactivates RNases which are present in virtually all biological materials and creates in combination with Buffer RB4 appropriate binding conditions which favor adsorption of RNA to the silica membrane A tedious and selective erythrocyte lysis as well as preparation of a leukocyte pellet is not necessary Contaminating DNA which is also bound to the silica membrane is removed by a recombinant DNase solution
7. 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 MACHEREY NAGEL 05 2014 Rev 02 27 RNA from blood 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 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 to 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 pr
8. in Buffer RB4 inhibits enzymatic reactions and has to be removed completely before eluting RNA Finally release the vacuum 8 Elute RNA Place the Elution Plate U bottom onto the spacers MTP Multi 96 Plate of the vacuum manifold Pipette 75 130 pL RNase free H O directly to the bottom of each well Incubate for 2 min at room temperature Build up the vacuum with the valve closed Once the maximum vacuum 0 6 bar is achieved open the valve and apply vacuum for 1 min Alternatively elution in standard PCR plates is possible Elution into PCR plates can be performed by placing a PCR plate onto a Square well Block resting on the spacers Square well Block in the manifold Reduction of atmospheric pressure MACHEREY NAGEL 05 2014 Rev 02 17 NucleoSpin 96 RNA Blood centrifuge processing 5 2 NucleoSpin 96 RNA Blood centrifuge processing For hardware requirements refer to section 2 3 For detailed information on each step see page 21 Before starting the preparation Check if Buffer RB3 Buffer RB4 and rDNase were prepared according to section 3 Protocol at a glance 1 Lyse blood 400 uL blood 400 pL DL 10 uL Liquid Proteinase K RT 15 min shake 1 000 1 200 rpm 2 Adjust binding conditions 400 pL RB4 Pipette up and down 10 15 times to mix 3 Transfer lysates to NucleoSpin RNA Blood Binding Plate 4 Bind RNA to silica membrane of the NucleoSpin R
9. required buffer volumes and pour an additional amount of 10 into the reservoirs Do not fill back unused buffer from reservoir into the bottle to avoid contaminations Ask technical service for extended buffer volumes 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 higher vacuum strength is required during the elution Transfer of sample solution to the NucleoSpin RNA Blood Binding Plate Be sure that no liquid drops out of the tips while moving the tips above the binding plate MACHEREY NAGEL 05 2014 Rev 02 25 RNA from blood 6 2 Ordering information Product REF Pack of NucleoSpin 96 RNA Blood 740225 2 2 x 96 preps 740225 4 4x 96 preps NucleoSpin 8 RNA Blood 740220 12 x 8 preps 740220 5 60 x 8 preps MN Square well Block 740476 4 740476 24 24 Round well Block Low 740482 4 Round well Block 740671 20 Rack of Tube Strips 740637 5 racks Cap Strips 740478 48 740478 24 288 Rack of Tube Strips 740477 4 sets 1 set consists of 1 rack 12 strips with 8 740477 24 24 sets tubes each and 12 Cap Strips MN Wash Plate 740479 4 740479 24 24 Elution Plate U bottom 740486 24 24 sets with Self adhering Foil Self adhering PE Foil 740676 50 NucleoVac 96 Vacuum Manifold 740681 1 NucleoVac Vacuum Regulator 740641 1 MN Frame 740680 1
10. well Block MACHEREY NAGEL 05 2014 Rev 02 21 NucleoSpin 96 RNA Blood centrifuge processing Dry silica membrane Residual wash buffer from the NucleoSpin RNA Blood Binding Plate is removed by the extended centrifugation time of 10 min after adding Wash Buffer RB4 described in the third washing step This prolonged time is necessary to eliminate any trace amounts of ethanol Note The ethanol in Buffer RB4 inhibits enzymatic reactions and has to be removed completely before eluting RNA Elute RNA For elution place the NucleoSpin RNA Blood Binding Plate onto a Round well block included in the kit and pipette 50 130 pL RNase free H O directly to the bottom of each well Make sure that all of the water comes into contact with the silica membrane and that the membrane is wet completely 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 see ordering information or standard PCR plates is possible For elution place the NucleoSpin RNA Blood Binding Plate on top of a MN Square well Block and centrifuge For direct elution onto PCR plates place a PCR plate between the NucleoSpin RNA Blood Binding Plate and the MN Square well Block before centrifugation Note The Elution Plate U bottom is not suitable for use in a centrifuge 22 MACHEREY NAGEL 05 2014 Rev 02 RNA from blood 6 Appendix 6 1 Troubleshooting
11. 014 Rev 02 NucleoSpin 96 RNA Blood vacuum processing Wash and dry silica membrane 500 pL RB2 0 2 bar 1 min 800 pL RB3 0 2 bar 1 min 500 pL RB4 0 2 bar 1 min Remove MN Wash Plate Dry silica membrane Maximum vacuum 10 min Elute RNA 75 130 pL RNase free H O RT 2 min 0 5 bar 1 min Reduction of atmospheric pressure MACHEREY NAGEL 05 2014 Rev 02 13 NucleoSpin 96 RNA Blood vacuum processing Setup of vacuum manifold Binding Washing Elution steps lt J NucleoSpin Binding Plate gt MN Wash Plate Elution Plate U bottom Manifold base with spacers Manifold base with spacers for MTP Multi 96 Plate inserted Microtube Rack inserted Binding Washing step Elution step 14 MACHEREY NAGEL 05 2014 Rev 02 NucleoSpin 96 RNA Blood vacuum processing Detailed protocol For vacuum processing of the NucleoSpin 96 RNA Blood kit all necessary consumables are included When processing a large number of samples under vacuum cross contamination is a major concern due to spraying of liquids or aerosol formation The MN Wash Plate prevents this contamination effected by droplets at the outlets of the individual wells of the NucleoSpin RNA Blood Binding Plate This consistent and effective tool is highly recommended for vacuum processing When using the NucleoSpin 96 RNA Blood kit under vacuum the Nuc
12. 20 C The frozen working solution is stable for 6 months Do not freeze thaw the aliquots more than three times Be careful when opening the vial as some particles of the lyophilisate may be attached to the lid rDNase reaction mixture For each sample to be processed mix 10 uL recon stituted rDNase with 90 pL Reaction Buffer for rDNase Wash Buffer RB3 Add the indicated volume of 96 100 ethanol see table below to Buffer RB3 Concentrate Mark the label of the bottle to indicate that ethanol was added Store Wash Buffer RB3 at room temperature 18 25 C for up to one year Wash Buffer RB4 Add the indicated volume of 96 100 ethanol see table below to Buffer RB4 Concentrate Mark the label of the bottle to indicate that ethanol was added Store Wash Buffer RB4 at room temperature 18 25 C for up to one year 8 MACHEREY NAGEL 05 2014 Rev 02 RNA from blood NucleoSpin 96 RNA Blood 12 x 8 preps 60 x 8 preps REF 740220 740220 5 rDNase RNase free 4 vials size D 8 vials size D lyophilized Add 540 uL Add 540 uL RNase free H O RNase free H O to each vial to each vial Wash Buffer RB3 100 mL 2x 100 mL Concentrate Add 400 mL ethanol Add 400 mL ethanol to each bottle Wash Buffer RB4 65 mL 2x65 mL Concentrate Add 150 mL ethanol Add 150 mL ethanol to each bottle MACHEREY NAGEL 05 2014 Rev 02 9 RNA from blood 4 Safety instructions The following components of the NucleoSpin 96 RN
13. A Blood kits contain hazardous contents Wear gloves 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 rDNase rDNase Iyophilized Warning 317 334 261 304 340 RNase free rDNase lyophilisiert Achtung 3424311 301 312 280 3024352 3334313 DL Guanidinium thiocyanate Warning 302 412 260 273 30 60 EUH031 301 312 330 Guanidiniumthiocyanat Achtung 30 60 RB2 Guanidinium thiocya Warning 226 302 210 233 nate 24 36 ethanol 301 312 330 20 35 403 235 Guanidiniumthiocyanat Achtung 24 36 Ethanol 20 35 Liquid Proteinase K liquid Danger 317 334 261 272 280 Proteinase K 1 10 302 352 Proteinase K fl ssig 1 10 Gefahr 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 asth
14. NA Blood Binding Plate 5 600 6 000 x g 2 min 5 Desalt silica membrane 6 Incubate with rDNase 500 pL RB3 5 600 6 000 x g 2 min 95 uL rDNase reaction mixture RT 15 min 18 MACHEREY NAGEL 05 2014 Rev 02 NucleoSpin 96 RNA Blood centrifuge processing Wash and dry silica membrane 500 pL RB2 5 600 6 000 x g 2 min 800 uL RB3 5 600 6 000 x g 2 min 500 pL RB4 5 600 6 000 x g 10 min Elute RNA 50 130 uL RNase free H O RT 2 min 5 600 6 000 x g 3 min MACHEREY NAGEL 05 2014 Rev 02 19 NucleoSpin 96 RNA Blood centrifuge processing Detailed protocol This standard protocol is recommended for purification of RNA from fresh or frozen whole blood stabilized with e g EDTA Na citrate or Li heparin Place the NucleoSpin RNA Blood Binding Plate on an MN Square well Block not supplied The use of a second plate placed on a MN Square well Block avoids the need to balance the centrifuge For waste collection suitable consumables e g MN Square well Blocks are necessary since they are not included in the kit For the most convenient handling without emptying and reuse of MN Square well Blocks we recommend to use six MN Square well Blocks if two 96 well plates are processed at once see ordering information Alternatively it is possible to empty the MN Square Blocks after every centrifugation step thus reducing the amount of MN Square well Block
15. NA contains a representative distribution of mRNAs blood samples should not be stored for long periods prior RNA isolation If long term storage of stabilized whole blood is necessary it is recommended to aliquot the blood samples and add the indicated volume of Lysis Buffer DL without adding Proteinase K Store the lysates at 20 C After thawing add Proteinase K MACHEREY NAGEL 05 2014 Rev 02 7 RNA from blood 3 Storage conditions and preparation of working solutions Attention Buffers DL and RB2 contain guanidinium thiocyanate Wear gloves and goggles Store lyophilized rDNase RNase free at 4 C on arrival stable up to 1 year All other kit components should be stored at room temperature 18 25 C and are stable for up to one year Storage at lower temperatures may cause salt precipitation If salt precipitation is observed incubate the bottle at 30 40 C for several minutes and mix well until all precipitates are redissolved After first use it is recommended to store Liquid Proteinase K at 4 C or 20 C Before starting any NucleoSpin 96 RNA Blood procedure prepare the following rDNase RNase free Add indicated volume of RNase free H O see table below to the rDNase vial and incubate for 1 min at room temperature Gently swirl the vials to completely dissolve the rDNase Be careful not to mix rDNase vigorously as rDNase is sensitive to mechanical agitation Dispense into aliquots and store at
16. Visit www mn net com for more detailed product information 26 MACHEREY NAGEL 05 2014 Rev 02 RNA from blood 6 3 Product use restriction warranty NucleoSpin 96 RNA Blood 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 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 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 N 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 VITRO diagnostic products are expressly marked as IVD on the packaging IF THERE IS NO IVD SIGN THE PRODUCT SHALL NOT BE SUITABLE FOR
17. d reusing the MN Square well Blocks we recommend using six MN Square well Blocks if two 96 well plates 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 2 4 Automated processing on robotic platforms NucleoSpin 96 RNA Blood can be readily automated on common laboratory robotic workstations For vacuum processing the use of the disposable MN Wash Plate inside the vacuum manifold is recommended The use of the MN Wash Plate reduces the risk of cross contamination caused by spraying of solutions during vacuum filtration steps 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 2 5 Handling preparation and storage of starting material NucleoSpin 96 RNA Blood kits are designed for isolating total RNA from fresh or fro zen whole blood Whole blood should be collected in the presence of an anticoagulant preferably EDTA Na citrate or Li heparin To obtain optimal results it is recommended processing blood samples within a few hours after collection when EDTA Na citrate or Li heparin collection tubes are used Samples should be stored at 4 C for no longer than 24 hours mRNAs derived from blood cells have different stabilities To ensure that the isolated R
18. eoSpin RNA Blood Binding Plate onto a new MN Square well Block not supplied 5 Desalt silica membrane Desalt the membrane by adding 500 uL Buffer RB3 to each well and centrifuge for 2 min at 5 600 6 000 x g 6 Incubate with rDNase Prepare rDNase reaction mixture as described in section 3 Leave the NucleoSpin RNA Blood Binding Plate on the MN Square well Block Pipette 95 uL rDNase reaction mixture directly to the bottom of each well of the NucleoSpin RNA Blood Binding Plate 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 7 Wash silica membrane Add 500 pL Buffer RB2 to each well of the NucleoSpin RNA Blood Binding Plate Place the NucleoSpin RNA Blood Binding Plate onto the MN Square well Block into the rotor bucket and centrifuge for 2 min at 5 600 6 000 x g Discard MN Square well Block Place NucleoSpin RNA Blood Binding Plate onto a new MN Square well Block not supplied Add 800 uL Buffer RB3 to each well of the NucleoSpin RNA Blood Binding Plate and centrifuge for 2 min at 5 600 6 000 x g Empty MN Square well Block Place NucleoSpin RNA Blood Binding Plate back onto the MN Square well Block Add 500 uL Buffer RB4 to each well of the NucleoSpin RNA Blood Binding Plate and centrifuge for or 10 min at 5 600 6 000 x g Discard MN Square
19. f DNA The probability of DNA detection with PCR increases with MACHEREY NAGEL 05 2014 Rev 02 5 RNA from blood 1 the number of DNA copies per preparation single copy target lt plastidial mitochondrial target lt plasmid transfected into cells 2 decreasing PCR amplicon size Kit specifications at a glance Parameter NucleoSpin 96 RNA Blood Format 96 well plate Processing Manual or automated vacuum or centrifugation Sample material lt 400 uL fresh or frozen whole blood e g stabilized with EDTA Na citrate or Li heparin Fragment size gt 200 nt Typical yield 7 ug 3 20 ug per 1 mL blood sample Aoso Asso 1 9 2 1 Elution volume 50 130 uL Preparation time 100 min plate Binding capacity 100 ug If smaller volumes than 400 uL blood are used adjust the volumes of Buffer DL and Buffer RB4 in step 1 and 2 of the corresponding protocol by maintaining the following ratio 1 1 1 sample Buffer DL Buffer RB4 Example 300 uL blood 300 uL Buffer DL 300 uL Buffer RB4 The volume of Liquid Proteinase K can be calculated as follows Blood volume uL 40 volume Proteinase K uL Example 300 uL blood 40 7 5 uL Liquid Proteinase K 2 3 Required hardware For an efficient lysis of the whole blood samples a suitable shaker is required e g Thermomixer Comfort with adapter plate for microtiterplates or deep well plate Eppendorf VARIOMAG TELESHAKER Thermo Scientific Vacuum processi
20. fen 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 A The symbol shown on labels refers to the precaution phrases of this section Das auf Etiketten dargestellte Symbol weist auf die P S tzen dieses Kapitels hin MACHEREY NAGEL 05 2014 Rev 02 11 NucleoSpin 96 RNA Blood vacuum processing 5 1 Protocols NucleoSpin 96 RNA Blood vacuum processing For hardware requirements refer to section 2 3 For detailed information regarding the vacuum manifold set up see page 15 For detailed information on each step see page 16 Before starting the preparation Check if Buffer RB3 Buffer RB4 and rDNase were prepared according to section 3 Protocol at a glance 1 Lyse blood 400 uL blood 400 pL DL 10 uL Liquid Proteinase K RT 15 min shake 1 000 1 200 rpm 2 Adjust binding conditions 400 uL RB4 Pipette up and down 10 15 times to mix 3 _ Transfer lysates to NucleoSpin RNA Blood Binding Plate 4 Bind RNA to silica membrane of the 0 2 bar 1 min NucleoSpin RNA Blood Binding Plate 5 Desalt silica membrane 500 pL RB3 0 2 bar 3 min 6 Incubate with rDNase 95 uL rDNase reaction mixture RT 15 min Reduction of atmospheric pressure 12 MACHEREY NAGEL 05 2
21. 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 28 MACHEREY NAGEL 05 2014 Rev 02
22. leoVac 96 Vacuum Manifold is required see ordering information Place NucleoSpin 96 RNA Blood Binding Plate on NucleoVac 96 Vacuum Manifold If processing less than 96 samples seal unused wells with a Self adhering PE Foil in order to ensure proper vacuum during the filtration steps This standard protocol is recommended for purification of RNA from fresh or frozen whole blood stabilized with e g EDTA Na citrate or Li heparin This standard protocol is recommended for purification of RNA from 400 uL fresh or frozen whole blood stabilized with for example EDTA Na citrate or Li heparin If smaller volumes than 400 uL blood are used adjust the volumes of Buffer DL and Buffer RB4 in step 1 and 2 according to section 2 2 ratio 1 1 1 sample Buffer DL Buffer RB4 Before starting the preparation Check if Buffer RB3 RB4 and rDNase were prepared according to section 3 1 Lyse blood Add 400 uL blood to each well of a Square well block Add 400 uL Buffer DL to each well Mix by shaking 1 000 1 200 rpm for 1 min For each blood sample add 10 uL Liquid Proteinase K Incubate for 15 min at room temperature on a shaker 1 000 1 200 rpm 2 Adjust binding conditions Add 400 uL Buffer RB4 to each sample Mix by pipetting up and down at least 10 15 times Optional Mix by shaking 1 000 rpm Note Buffer DL and Buffer RB4 have to be used in the same volume ratio MACHEREY NAGEL 05 2014 Rev 02 15 NucleoS
23. maartige Symptome oder Atembeschwerden verursachen H 412 Harmful to aquatic life with long lasting effects Sch dlich f r Wasserorganismen mit langfristiger Wirkung EUH 031 Contact with acids liberates toxic gas Entwickelt bei Ber hrung mit S ure giftige Gase 10 MACHEREY NAGEL 05 2014 Rev 02 RNA from blood Precaution phrases P 261 P 272 P 280 P 301 312 P 302 352 P 304 340 P 333 313 P 342 311 P 363 Avoid breathing dust Einatmen von Staub vermeiden Contaminated work clothing should not be allowed out of the workplace Kontaminierte Arbeitskleidung nicht auBerhalb des Arbeitsplatzes tragen 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 If breathing is difficult remove to fresh air and keep at rest ina position comfortable for breathing BEI EINATMEN An die frische Luft bringen und in einer Position ruhigstellen die das Atmen erleichtert If skin irritation occurs Get medical advice attention Bei Hautreizung Arztlichen Rat einholen arztliche Hilfe hinzuziehen If experiencing respiratory symptoms Call a POISON CENTER doctor Bei Symptomen der Atemwege GIFTINFORMATIONSZENTRUM Arzt anru
24. ng The NucleoSpin 96 RNA Blood kit can be used with either the NucleoVac 96 Vacuum Manifold see ordering information When using NucleoSpin 96 RNA Blood with less than 96 samples Self adhering PE Foil see ordering information should be used in order to close and protect non used wells of the NucleoSpin 96 RNA Blood Binding Plate and thus guarantee a proper vacuum 6 MACHEREY NAGEL 05 2014 Rev 02 RNA from blood Establish a reliable vacuum source for the NucleoVac 96 Vacuum Manifold The manifold may be used with a vacuum pump house 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 Centrifugation For centrifugation a microtiterplate centrifuge is required This centrifuge must be able to accommodate the NucleoSpin RNA Blood Binding Plate stacked on a round or square well block 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 an
25. oduct 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 Last updated 07 2010 Rev 03 Please contact MACHEREY NAGEL GmbH amp Co KG Tel 49 24 21 969 270 tech bio mn net com Trademarks Disclaimer NucleoSpin is a registered trademark of MACHEREY NAGEL GmbH amp Co KG VARIOMAG is a registered trademark of Thermo Scientific Inc 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
26. pin 96 RNA Blood vacuum processing Prepare NucleoVac 96 Vacuum Manifold Insert spacers MTP Multi 96 Plate notched side up into the grooves located on the short sides of the manifold Insert the waste reservoir into the center of the manifold Place the MN Wash Plate on the spacers in the manifold base 3 Transfer lysates to NucleoSpin RNA Blood Binding Plate Place a NucleoSpin RNA Blood Binding Plate into vacuum manifold s lid and apply the samples to the wells 4 Bind RNA to silica membrane Apply vacuum until all lysates have passed through the wells 0 2 bar 1 min Release the vacuum 5 Desalt silica membrane Desalt the membrane by adding 500 uL Buffer RB3 to each well and apply vacuum 0 2 bar 3 min until all buffer has passed through the wells Release the vacuum 6 Incubate with rDNase Prepare rDNase reaction mixture as described in section 3 Pipette 95 pL rDNase reaction mixture directly to the bottom of each well in the NucleoSpin RNA Blood Binding Plate 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 comes into contact with the silica membrane and that the membrane is wet completely 7 Wash silica membrane Add 500 pL Buffer RB2 to each well of the NucleoSpin RNA Blood Binding Plate Apply vacuum 0 2 bar 1 min until all buffer has passed through the wells Release the vacuum
27. s needed This standard protocol is recommended for purification of RNA from 400 uL fresh or frozen whole blood stabilized with for example EDTA Na citrate or Li heparin If smaller volumes than 400 uL blood are used adjust the volumes of Buffer DL and Buffer RB4 in step 1 and 2 according to section 2 2 ratio 1 1 1 sample Buffer DL Buffer RB4 Before starting the preparation Check if Buffer RB3 RB4 and rDNase were prepared according to section 3 1 Lyse blood Add 400 uL blood to each well of a Square well block included Add 400 uL Buffer DL to each well Mix by shaking 1 000 1 200 rpm for 1 min For each blood sample add 10 uL Liquid Proteinase K Incubate for 15 min at room temperature on a shaker 1 000 1 200 rpm 2 Adjust binding conditions Add 400 uL Buffer RB4 to each sample Mix by pipetting up and down at least 10 15 times Optional Mix by shaking 1 000 rpm Note Buffer DL and Buffer RB4 have to be used in the same volume ratio 3 Transfer lysates to NucleoSpin RNA Blood Binding Plate Place the NucleoSpin RNA Blood Binding Plate on an MN Square well Block not supplied and transfer lysates into the wells of the NucleoSpin RNA Blood Binding Plate 20 MACHEREY NAGEL 05 2014 Rev 02 NucleoSpin 96 RNA Blood centrifuge processing 4 Bind RNA to silica membrane Centrifuge for 2 min at 5 600 6 000 x g Discard MN Square well Block with flow through and place Nucl
28. supplied The recombinant DNase solution is directly applied onto the silica membrane during the preparation Simple washing steps with two different buffers remove salts metabolites and macromolecular cellular components Finally the pure RNA is eluted under low ionic strength conditions with RNase free H O supplied 2 2 Kit specifications The NucleoSpin 96 RNA Blood kits are recommended for the isolation of RNA from fresh or frozen whole blood e g stabilized with EDTA Na citrate or Li heparin The NucleoSpin 96 RNA Blood kits can be used on fully automated common laboratory workstations See section 2 4 The NucleoSpin 96 RNA Blood kits can be used manually under vacuum or under centrifugation For use under centrifugation additional consumables for waste collection e g MN Square well Blocks have to be ordered separately Please see section 2 3 for further details The NucleoSpin 96 RNA Blood kits allow the purification of RNA with an Azeo Azgo ratio typically exceeding 1 9 The isolated RNA is ready to use for typical downstream applications e g reverse transcriptase PCR RT PCR RNA isolated with the NucleoSpin 96 RNA Blood kit is typically of high integrity However RNA integrity strongly depends on the sample quality The amount of DNA contamination is significantly reduced during on column digestion with rDNase However in very sensitive applications it may be possible to detect traces o
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