Total RNA Isolation
Contents
1. MACHEREY NAGEL 01 2010 Rev 02 9 Total RNA Isolation 3 Storage conditions and preparation of working solutions Attention Buffers MR1 and MR3 contain chaotropic salt Wear gloves and goggles All components of the NucleoMag 96 RNA kit should be stored at room tem perature 20 25 C and are stable for up to one year All buffers are delivered ready to use Before starting NucleoMag 96 RNA protocol prepare the following rDNase working solution Add 800 ul of RNase free H O to each 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 sen sitive to mechanical agitation If not used completely this working solution can be stored at 20 C for up to 6 month Do not freeze thaw the rDNase working solution more than three times rDNase reaction mixture Add 9 2 ml Reaction Buffer for rDNase to 800 ul rDNase working solution and mix The resulting rDNase reaction mixture will be sufficient for 32 isolations and should be used up When performing less than 32 reactions prepare a smaller amount of the reaction mixture For each isola tion combine 276 ul of reaction buffer for rDNase with 24 ul of rDNase working solution Reducing Agent TCEP Add 750 ul of RNase free H O to the TCEP vial and incubate for several minutes at room temperature Mix the vial to dissolve the TCEP completely Store dissolved TCEP at 22. 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 state ments 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 MACHEREY NAGEL 01 2010 Rev 02 21 Total RNA Isolation 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 Please contact MACHEREY NAGEL Germany Tel 49 0 24 21 969 270 e mail TECH BIO mn net com Last updated 12 2006 Rev 02 Trademarks KingFisher is a registered trademark of Thermo Fisher Scientific NucleoMag is a trademark of MACHEREY NAGEL GmbH amp Co KG
3. Total RNA Isolation User Manual NucleoMag 96 RNA January 2010 Rev 02 MACHEREY NAGEL MN Total RNA Isolation Table of contents 1 Components 1 1 Kit contents 1 2 Material to be supplied by user 2 Product description 2 1 The basic principle 2 2 Kit specifications 2 3 Magnetic separation systems 2 4 Adjusting the shaker settings 2 5 Handling of beads 2 6 Elution procedures 3 Storage conditions and preparation of working solutions 4 Safety instructions risk and safety phrases 5 Procedure 5 1 General procedure 5 2 Protocol for the isolation of total RNA from cells or tissue 6 Appendix 6 1 Troubleshooting 6 2 Ordering information 6 3 Product use restriction warranty a A Oo woOoON DD DD 10 11 13 13 15 18 18 20 21 MACHEREY NAGEL 01 2010 Rev 02 Total RNA Isolation 1 Components 1 1 Kit contents 1x 96 preps 744350 1 NucleoMag 96 RNA 4 x 96 preps 744350 4 NucleoMag B Beads 3 ml 12 ml Lysis Buffer MR1 50 ml 200 ml Binding Buffer MR2 80 ml 320 ml Wash Buffer MR3 80 ml 320 ml Wash Buffer MR4 250 ml 1000 ml Elution Buffer MR5 25 ml 100 ml Reducing Agent TCEP 1 vial 4 vials rDNase lyophilized Reaction Buffer for rDNase RNase free H O User Manual 107 mg vial 3 vials size D 35 ml 5 ml 1 107 mg vial 12 vials size D 4x 35 ml 20 ml 1 For preparation of working solutions and storage conditions see section 3
4. Elution Buffer MR5 RNase free water 4 MACHEREY NAGEL 01 2010 Rev 02 Total RNA Isolation 1 2 Material to be supplied by user Product Cat No Pack of Separation plate for magnetic beads separa tion 740481 4 e g Square well Block 96 well block with 740481 24 24 2 1 ml square wells Lysis tubes for incubation of samples and lysis e g Rack of Tubes Strips 1 set consists of 1 Rack 12 Strips with 8 tubes 1 2 ml wells each and 12 Cap Strips 740477 4 4 sets 740477 24 24 sets Elution plate for collecting purified RNA e g Elution Plate U bottom 96 well 0 3 ml 740486 24 24 microtiterplate with 300 ul u bottom wells e g Elution Plate Flat bottom 96 well 740673 20 0 3 ml microtiterplate with 300 ul flat bottom wells For use of kit on KingFisher 96 instrument KingFisher 96 Accessory Kit B Square well Blocks Deep well tip combs 740951 1 set Elution Plates for 4 x 96 NucleoMag 96 RNA preps using KingFisher 96 platform MACHEREY NAGEL 01 2010 Rev 02 Total RNA Isolation 2 Product description 2 1 The basic principle The NucleoMag 96 RNA procedure is based on the reversible adsorption of nucleic acids to paramagnetic beads under appropriate buffer conditions Sample lysis is achieved by homogenization in a solution containing chaotropic ions For the adjustment of conditions under which nucleic acids bind to the paramagnetic beads Buffer MR2 and the NucleoMag B Bead
5. and the volume to be processed The individual times for complete attraction of the beads to the magnetic pins should be checked and adjusted on each system It is recommended to use the separation plates or tubes specified by the supplier of the magnetic separator Washing the beads Washing the beads can be achieved by shaking or mixing In contrast to mixing by pipetting up and down mixing by shaker or magnetic mixing allows simultaneous mixing of all samples This reduces the time and number of tips needed for the preparation Resuspension by pipetting up and down however is more efficient than mixing by a shaker or magnetic mix 8 MACHEREY NAGEL 01 2010 Rev 02 Total RNA Isolation Method Resuspension Speed Number of tips efficiency needed Magnetic mix Shaker Pipetting 8 channel pipetting device 2 6 Elution procedures Purified total RNA can be eluted directly with the supplied Elution Buffer MR5 Elution can be carried out in a volume of 50 ul It is essential to cover the NucleoMag B Beads completely with Elution Buffer MR5 during the elution step The volume of dispensed Elution Buffer MR5 depends on the magnetic separation system e g the position of the pellet inside the separation plate For efficient elution the magnetic bead pellet should be resuspended completely in the Elution Buffer MR5 For some separators high elution volumes might be necessary to cover the whole magnetic bead pellet
6. in less than 120 minutes using the NucleoMag SEP on automation platforms The kit provides reagents for the purification of up to 30 ug of pure RNA from suitable samples Depending on the elution volume used concentrations of 10 30 ng l can be obtained NucleoMag 96 RNA can be processed completely at room temperature NucleoMag B Beads are highly reactive superparamagnetic beads The binding capac ity is approx 1 ug of RNA per 1 ul of NucleoMag B Bead Suspension 1 ul of suspen sion contains 130 ug beads Elution Buffer MR5 RNase free water 6 MACHEREY NAGEL 01 2010 Rev 02 Total RNA Isolation 2 3 Magnetic separation systems For use of NucleoMag 96 RNA the use of the magnetic separator NucleoMag SEP is recommended Separation is carried out in a Square well Block see ordering informa tion The kit can also be used with other common separators See suppliers ordering information for suitable separation plates Magnetic separator Separation plate or tube NucleoMag SEP MN Cat No 744900 Square well Block MN Cat No 740670 Tecan Te MagS 1 5 ml tubes without lid Sarstedt Static magnetic pins Separators with static magnetic pins for example NucleoMag SEP for manual use and for use on liquid handling workstations This type of separator is recommended in combination with a suitable microplate shaker for optimal resuspension of the beads during the washing and elution steps Alternative
7. 0 C NucleoMag 96 RNA 1 x 96 preps 4x 96 preps Cat No 744350 1 744350 4 rDNase lyophilized 3 vials size D 12 vials size D Add 800 ul RNase free H O Add 800 ul RNase free H O to each vial to each vial TCEP 1 vial 107 mg 4 vials 107 mg vial Add 750 ul RNase free H O Add 750 pl RNase free H O to each vial 10 MACHEREY NAGEL 01 2010 Rev 02 Total RNA Isolation 4 Safety instructions risk and safety phrases The following components of the NucleoMag 96 RNA kits contain hazardous con tents Wear gloves and goggles and follow the safety instructions given in this section Component Hazard contents Hazard Risk phrases Safety phrases MR1 Guanidine x Xn Harmful by inhalation R S13 thiocyanate in contact with the 20 21 22 skin and if swallowed MR2 lsopropanol 4 F Highly flammable R 11 36 S 7 16 gt 90 i Irritating to eyes 67 24 25 26 Vapours may cause 3 Xn drowsiness and diz ziness MR3 Guanidine x Xn Flammable Harmful R 10 S 13 16 thiocyanate by inhalation in 20 21 22 ethanol lt 40 contact with the skin and if swallowed MR4 Ethanol lt 80 pe Highly flammable R11 S 7 16 rDNase rDNase x Xi May cause sensitiza R 42 43 S 22 24 RNase free lyophilized tion by inhalation and skin contact Reducing Tris 2 car x Xi Causes burns R 34 S 26 27 Agent TCEP boxyleth 36 37 39 ylphosphine Hydrochloride Risk phrases R 10 Fl
8. Blocks 740481 4 4 740481 24 24 Self adhering PE Foil 740676 50 sheets Rack of Tube Strips 740477 4 4 sets set consists of 1 Rack 12 Tube Strips 740477 24 24 sets with 8 tubes each and Cap Strips Cap Strips 740638 30 strips KingFisher 96 Accessory Kit B 744951 1 set Square well Blocks Deep well tip combs Elution Plates for 4 x 96 NucleoMag 96 RNA preps using KingFisher 96 platform Visit www mn net com for more detailed product information 20 MACHEREY NAGEL 01 2010 Rev 02 Total RNA Isolation 6 3 Product use restriction warranty NucleoMag 96 RNA kit components were developed designed distributed and sold FOR RESEARCH PURPOSES ONLY They are suitable FOR IN VITRO USES ONLY No claim or representation is intended for its use to identify any specific organism or for Clinical use diagnostic prognostic therapeutic or blood banking It is rather the responsibility of the user to verify the use of the NucleoMag 96 RNA kit for a specific application range as the performance characteristic of this kit has not been verified to a specific organism This MACHEREY NAGEL product is shipped with documentation stating specifica tions 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 ma
9. Te MagS is a trademark of Tecan Group Ltd 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 22 MACHEREY NAGEL 01 2010 Rev 02
10. ammable R11 Highly flammable R 20 21 22 Harmful if swallowed R 34 Causes burns R 36 Irritating to eyes R 42 43 May cause sensitization by inhalation and skin contact R 67 Vapours may cause drowsiness and dizziness Hazard labeling not necessary 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 Hazard labeling not necessary if quantity per bottle below 25g 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 MACHEREY NAGEL 01 2010 Rev 02 11 Total RNA Isolation Safety phrases S7 S13 S16 S22 S24 S 24 25 S 26 S 27 S 36 37 38 Keep container tightly closed Keep away from food drink and animal feedstuffs Keep away from sources of ignition No smoking Do not breathe dust Avoid contact with the skin Avoid contact with skin and eyes In case of contact with eyes rinse immediately with plenty of water and seek medical advice Take off immediately all contaminated clothing Wear suitable protective clothing and gloves 12 MACHEREY NAGEL 01 2010 Rev 02 NucleoMag 96 RNA 5 Procedure 5 1 General procedure 1 Homogenize lyse Up to 20 mg tissue sample or 2 x 10 cells 350 p
11. de to the general business terms and conditions of MACHEREY NAGEL which are printed on the price list Please contact us if you wish an extra copy MACHEREY NAGEL does not warrant against damages or defects arising in shipping and handling transport insurance for customers excluded or out of accident or im proper or abnormal use of this product against defects in products or components not manufactured by MACHEREY NAGEL or against 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 spe cial 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
12. eads are resuspended completely during the washing procedure If shaking is not sufficient to resuspend the beads completely mix by repeated pipetting up and down 18 MACHEREY NAGEL 01 2010 Rev 02 Total RNA Isolation Problem Possible cause and suggestions Carry over of ethanol from wash buffers i Or Par e Be sure to remove all of the ethanolic wash solution as residual ormance ethanol interferes with downstream applications of RNA in downstream L it applications OW PENY e See above Time for magnetic separation too short e Increase separation time to allow the beads to be completely attracted to the magnetic pins before aspirating any liquid from Carry over the well of beads Aspiration speed too high elution step e High aspiration speed during the elution step may cause bead carry over Reduce aspiration speed for elution step Contamination of the rims Cross con e Donot moisten the rims of the Square well Block when transfer tamination ring the tissue lysate If the rim of the wells is contaminated seal the Square well Block with Self adhering PE Foil see ordering information before starting the shaker MACHEREY NAGEL 01 2010 Rev 02 19 Total RNA Isolation 6 2 Ordering information Product Cat No Pack of NucleoMag 96 RNA 744350 1 1 x 96 preps 744350 4 4 x 96 preps NucleoSpin Filters 740606 50 NucleoSpin RNA Filter Plates 740711 4 NucleoMag SEP 744900 1 Square well
13. he kit to robotic instruments 1 Homogenize lyse sample Lyse up to 20 mg of tissue or 2 x 10 cells in 350 pl Buffer MR1 For tissue samples Use a suitable homogenization tool to homogenize samples in Buffer MR1 Samples can be disrupted using bead based homogenization tools for example GenoGrinder or Mixer Mill MM400 see instrument manu facturer s recommendations for suitable plates or tubes for homogenization or any other suitable homogenization tools Centrifuge the crude homogenate to pellet debris or remaining tissue particles Alternatively NucleoSpin RNA Filter Tubes or Plates can be used to clear the crude lysate see ordering informa tion Transfer the clear supernatant to the Square well Block see ordering information for further processing For cells Add Buffer MR1 to cell pellet Pipette up and down several times to lyse the cells Optionally Use NucleoSpin RNA Filter Tubes or Plates see ordering information or a syringe to reduce the viscosity of the lysate Transfer lysate to the Square well Block for further processing 2 Bind RNA to NucleoMag B Beads Add 28 pl resuspended NucleoMag B Beads and 350 ul Buffer MR2 to the lysed sample Mix by pipetting up and down 6 times and incubate for 5 min at room temperature NucleoMag B Beads and Buffer MR2 can be premixed Be sure to resuspend the NucleoMag B Beads before removing them from the storage bottle Vortex storage bottle briefly until a homogenous s
14. l MR1 6 pl TCEP Mix or use mechanical disruption 2 Bind RNA to 28 ul B Beads NucleoMag B Beads 350 pl MR2 Mix Incubate 5 min at RT Optional Mix by shaking Remove supernatant after 2 min separation Dry for 5 min at RT 3 rDNase digestion 300 pl rDNase reaction mixture Mix Incubate 15 min at RT 4 Rebinding 350 pl MR2 Mix Incubate 5 min at RT Remove supernatant after 2 min separation MACHEREY NAGEL 01 2010 Rev 02 13 NucleoMag 96 RNA 5 MR3 wash 600 pl MR3 Resuspend separate 2 min Aspirate and discard supernatant 6 MR4 wash 900 pl MR4 Resuspend separate 2 min Aspirate and discard supernatant Repeat washing step once 7 Drying Incubate for 10 15 min at RT 8 Elution 50 200 pl MR5 Shake 5 10 min RT Optional Mix by pipetting up and down Transfer eluted RNA after 2 min separation 14 MACHEREY NAGEL 01 2010 Rev 02 NucleoMag 96 RNA 5 2 Protocol for the isolation of total RNA from cells or tissue This protocol is designed for magnetic separators with static pins e g NucleoMag SEP and suitable plate shakers It is recommended using a Square well Block for separation see ordering information Alternatively isolation of RNA can be performed in reaction tubes with suitable magnetic separators This protocol is for manual use and serves as a guideline for adapting t
15. ly beads can be resuspended in the buffer by pipetting up and down several times For fully automated use on liquid handling workstations a gripper tool is required the plate is transferred to the magnetic separator for separation of the beads and transferred to the shaker module for resus pension of the beads Movable magnetic systems Separators with moving magnetic pins for example Te MagS for automated use only Magnetic pins rods are moved from one side of the well to the other and vice versa Beads follow this movement and are thus pulled through the buffer during the wash and elution steps Separation takes place when the system stops Automated separators Separators with moving magnets for example Thermo Fisher Scientific KingFisher instruments Magnetic beads are transferred into suitable plates or tubes Beads are resuspended from the rod covered magnets Following binding washing or elution beads are collected again with the rod covered magnets and transferred to the next plate or tube MACHEREY NAGEL 01 2010 Rev 02 7 Total RNA Isolation 2 4 Adjusting the shaker settings When using a plate shaker for the washing and elution steps the speed settings have to be adjusted carefully for each specific separation plate and shaker to prevent cross contamination from well to well Proceed as follows Adjusting shaker speed for wash steps e Load 900 ul dyed water to the wells of the separation plate Place
16. nt containing the purified total RNA to a suitable collection plate see ordering information MACHEREY NAGEL 01 2010 Rev 02 17 Total RNA Isolation 6 Appendix 6 1 Troubleshooting Problem Possible cause and suggestions RNase contamination RNA is e Create an RNase free working environment Wear gloves degraded during all steps of the procedure Change gloves frequently no RNA Use of sterile disposable polypropylene tubes or plates is obtained recommended Glassware should be oven baked for at least 2 hat 250 C before use Elution buffer volume insufficient e Beads pellet must be covered completely with elution buffer Insufficient performance of elution buffer during elution step e Remove residual buffers during the separation steps complete ly Remaining buffers decrease the efficiency of following wash and elution steps Beads dried out Poor RNA e Do not let the beads dry as this might result in lower elution ef yield ficiencies Aspiration of attracted bead pellet e Do not disturb the attracted beads while aspirating the super natant especially when the magnetic bead pellet is not visible in the lysate Aspiration and loss of beads e Time for magnetic separation too short or aspiration speed too high Insufficient washing procedure e Use only the appropriate combinations of separator and plate for example Square well Block in combination with NucleoMag Low purity SEP e Make sure that b
17. or Add 600 pl Buffer MR3 resuspend the beads by pipetting up and down Incubate for 1 min Separate the magnetic beads by placing the Square well Block on the NucleoMag SEP magnetic separator Wait at least 2 min until all the beads have been attracted to the magnets Remove and discard supernatant by pipet ting MR4 wash Remove the Square well Block from the NucleoMag SEP magnetic separator Add 900 ul Buffer MR4 and resuspend the beads by pipetting up and down Incubate for 1 min Separate the magnetic beads by placing the Square well Block on the NucleoMag SEP magnetic separator Wait at least 2 min until all the beads have been attracted to the magnets Remove and discard supernatant by pipet ting Repeat washing step once with 900 ul Buffer MR4 Leave the Square well Block on the NucleoMag SEP magnetic separator for the following step 16 MACHEREY NAGEL 01 2010 Rev 02 NucleoMag 96 RNA Drying Air dry the beads for 10 15 min at room temperature Elution Remove the Square well Block from the NucleoMag SEP magnetic separator Add desired volume of Buffer MR5 at least 50 pl 50 200 pl and resuspend the beads by pipetting up and down Incubate the suspension for 5 min at room temperature Separate the magnetic beads by placing the Square well Block on the NucleoMag SEP magnetic separator Wait at least 2 min until all the beads have been attracted to the magnets Transfer the supernata
18. s are added to the lysate After magnetic separation the paramagnetic beads are incubated with a recombinant DNase to remove co purified DNA Following a RNA rebinding step contaminants and salts are removed using the Wash Buffers MR3 and MR4 Residual ethanol from previous wash steps is removed by air drying Finally highly pure RNA is eluted with Elution Buffer MR5 and the RNA can directly be used for downstream applications The NucleoMag 96 RNA kit can be used either manually or automated on standard liquid handling instruments or auto mated magnetic separators 2 2 Kit specifications NucleoMag 96 RNA is designed for rapid manual and automated small scale prepara tion of highly pure total RNA from tissue or cell samples The kit is designed for use with NucleoMag SEP magnetic separator plate see ordering information or other magnetic separation systems see section 2 3 Manual time for the preparation of 96 samples is about 120 minutes The purified RNA can be used directly as template for RT PCR or any kind of enzymatic reactions Due to the recombinant DNase provided with the kit eluted RNA is virtually DNA free NucleoMag 96 RNA allows easy automation on common liquid handling instruments or automated magnetic separators for example Thermo Fisher Scientific KingFisher instruments The actual processing time depends on the configuration of the instrument and the magnetic separation system used Typically 96 samples can be purified
19. the plate on the shaker and start shaking with a moderate speed setting for 30 seconds Turn off the shaker and check plate surface for small droplets of dyed water e Increase speed setting shake for an additional 30 seconds and check plate surface for droplets again e Continue increasing the speed setting until you observe droplets on top of the separation plate Reduce speed setting check again and use this setting for the washing step Adjusting shaker speed for the elution step e Load 100 ul dyed water to the wells of the collection plate and proceed as de scribed above 2 5 Handling of beads Distribution of beads Ahomogeneous distribution of the magnetic beads to the individual wells of the separa tion plate is essential for a high well to well consistency Therefore before distributing the beads make sure that the beads are completely resuspended Shake the storage bottle well or place it on a vortexer shortly Premixing magnetic beads with the binding buffer allows easier homogenous distribution of the beads to the individual wells of the separation plate During automation a premix step before aspirating the beads binding buffer mixture from the reservoir is recommended to keep the beads resuspended Magnetic separation time Attraction of the magnetic beads to the magnetic pins depends on the magnetic strength of the magnetic pins the selected separation plate distance of the separation plate from the magnetic pins
20. uspension has formed Separate the magnetic beads against the side of the wells by placing the Square well Block on the NucleoMag SEP magnetic separator Wait at least 2 min until all the beads have been attracted to the magnets Remove and discard super natant by pipetting Remove supernatant completely Do not disturb the attracted beads while aspirating the supernatant GenoGrinder http www spexcsp com sampleprep Mixer Mill MM400 http www retsch com products milling ball mills mm 400 MACHEREY NAGEL 01 2010 Rev 02 15 NucleoMag 96 RNA Dry beads for 5 min at room temperature Keep the Square well Block on the NucleoMag SEP magnetic separator for the drying step rDNase digestion Remove the Square well Block from the NucleoMag SEP magnetic separator Add 300 pl rDNase reaction mixture and resuspend the beads by pipetting up and down Incubate for 15 min at room temperature Do not separate the beads Following incubation proceed with step 4 Rebinding Add 350 pl Buffer MR2 to each sample Mix by pipetting up and down 6 times and incubate for 5 min at room temperature Separate the magnetic beads against the side of the wells by placing the Square well Block on NucleoMag Sep magnetic separator Wait at least 2 min until all the beads have been attracted to the magnets Remove and discard supernatant by pipetting MR3 wash Remove the Square well Block from the NucleoMag SEP magnetic separa t
Download Pdf Manuals
Related Search