ChargeSwitch® Total RNA Cell Kits
Contents
1. Not intended for any animal or human therapeutic or diagnostic use invitrogen Corporate Headquarters Invi Corporation 1600 Faraday Avenue Carlsbad CA 92008 T 1 760 603 7200 F 1 760 602 6500 E tech service invitrogen com For country ific contact information visit our web site at www invitrogen com2. RNA CS14010 50 Cell Kit ChargeSwitch Total RNA CS14010S 10 Cell Kit Sample Size All components of the ChargeSwitch Total RNA Cell Kits except the DNase I shipped separately in dry ice are shipped at room temperature Upon receipt store components as follows e Store Proteinase K and Lysis Buffer L16 at 4 C e Store DNase I at 20 C e Store the remaining components at room temperature All components are guaranteed stable for 6 months when stored properly Continued on next page Kit Contents and Storage Continued Contents The components supplied in the ChargeSwitch Total RNA Kits are listed below Note Some reagents in the kit may be provided in excess of the amount needed Component Amount CS14010 CS14010S ChargeSwitch Magnetic Beads 25 mg ml in 1 mM 5 ml 1ml sodium acetate pH 4 5 Proteinase K 20 mg ml in 50 mM Tris HCI pH 8 5 250 ul 250 ul 50 glycerol and 5 mM CaCl2 DNase I 1 U l in 20 mM sodium acetate pH 6 5 250 ul 250 ul 5 mM CaCh 0 1 mM PMSF 50 glycerol DNase I Buffer 40 mM Tris HCI pH 8 0 12 5 ml 12 5 ml 10 mM MgsOz 3 mM CaCl Lysis Buffer L16 25 ml 25 ml Binding Buffer B9 14 ml 14 ml Wash Buffer W13 37 5 ml 37 5 ml Wash Buffer W14 50 ml 50 ml Elution Buffer E7 10 mM Tris HCl pH 9 0 7 5 ml 7 5 ml vi Product Qualification Product Qualification Each kit is functionally tested to ensure conforman
3. a 1 ml adjustable pipette tip set to 700 ul to mix the sample without forming bubbles Important Avoid forming bubbles by ensuring that the pipette tip is submerged during mixing and by pipetting up and down gently Incubate at room temperature for 1 minute to allow the RNA to bind to the beads Place the sample on the MagnaRack until the beads have formed a tight pellet and the supernatant is clear Without removing the tube from the magnet carefully aspirate and discard the supernatant without disturbing the pellet of beads by angling the pipette such that the tip is pointed away from the pellet see figure on page 5 If DNA free RNA is required proceed to DNase I Treatment next page If your downstream application tolerates the presence of genomic DNA proceed to Washing RNA page 14 Note The DNase I digestion can also be performed on the purified total RNA but this usually decreases the RNA yield 12 Continued on next page Isolating Total RNA from Cells Continued DNase Treatment Perform DNase I Treatment to prepare DNA free RNA 1 10 11 Remove the tube containing the pelleted magnetic beads from the magnet There should be no supernatant in the tube Add 500 ul Wash Buffer W14 to the tube and pipet up and down gently 5 times to resuspend the magnetic beads using a 1 ml pipette tip set to 700 ul to mix the sample without forming bubbles Place the sample on the magnet until the bea
4. set at 60 C e Ice Components Supplied with the Kit e ChargeSwitch Lysis Buffer L16 e Proteinase K Lysis Mix For each sample mix the following items in a microcentrifuge tube e 500 pl of ChargeSwitch Lysis Buffer L16 e 5 ulfresh 0 5 M DTT e 5 ulof Proteinase K For multiple samples prepare a master Lysis Mix by scaling up the volume of reagents accordingly Continued on next page Sample Preparation Continued Preparing Mammalian Cell Lysates cS oy D a Remove the growth medium from the cells Note Incomplete removal of the growth medium will inhibit the lysis and affect the efficiency of the RNA purification Adherent cells up to 1 x 10 cells Completely remove the growth medium from the monolayer Proceed with Step 2 Note Depending on the cell type you may use trypsin or a rubber policeman to remove all cells from the culture plate Transfer the detached cells to a microcentrifuge tube and centrifuge briefly depending on cell type to pellet the cells Completely remove the growth medium and proceed with Step 2 Suspension cells up to 1 x 10 cells Transfer the cells to a microcentrifuge tube and centrifuge briefly depending on cell type to pellet cells Completely remove the growth medium Proceed with Step 2 Wash the cells with 1X PBS Centrifuge detached cells or suspension cells by centrifugation Completely remove PBS from cells Resuspend the cells in 500 ul of th
5. Buffer is lower than the volume of beads used RNA elution is incomplete You may need to perform a second elution to recover all RNA Elution Buffer Temperature Prewarming the elution buffer to 60 70 C increases the RNA yield Continued on next page General Information Continued Safety Follow the safety guidelines below when using the Information ChargeSwitch Total RNA Kits Treat all reagents supplied in the kit as potential irritants Always wear a suitable lab coat disposable gloves and protective goggles If a spill of the buffers occurs clean with a suitable laboratory detergent and water If the liquid spill contains potentially infectious agents clean the affected area first with laboratory detergent and water then with 1 v v sodium hypochlorite or a suitable laboratory disinfectant Sample Preparation Introduction Materials Needed Preparing Reagents Instructions for isolating total RNA from 1 x 10 mammalian cells or 1 ml overnight bacterial culture are described below Read the entire instructions before starting the purification procedure You will need the following items e Sample up to 1 x 10 mammalian cells or 1 ml overnight bacterial culture ODeo 0 6 to 1 2 e Sterile 1 5 ml microcentrifuge tubes e Adjustable pipettes and aerosol barrier pipette tips e 1X Phosphate Buffered Saline PBS page viii e Fresh 0 5 M DTT RNase free e Water bath or heat block
6. DNA does not affect downstream applications proceed directly with the washing step 1 Remove the tube containing the pelleted magnetic beads from the magnet Step 6 page 12 There should be no supernatant in the tube Add 750 ul Wash Buffer W13 to the tube Resuspend the magnetic beads by pipeting up and down gently at least 5 times using a 1 ml pipette tip set to 700 ul to mix the sample without forming bubbles or vortex at low speed to avoid any solution from collecting in the tube cap Place the sample on the magnet until the beads have formed a tight pellet and the supernatant is clear Without removing the tube from the magnet carefully aspirate and discard the supernatant without disturbing the pellet by angling the pipette such that the tip is pointed away from the pellet Remove the tube from the magnet Add 750 ul Wash Buffer W14 or 500 ul if you performed DNase I treatment to the tube Resuspend the magnetic beads by pipeting up and down gently 3 times to using a 1 ml adjustable pipette tip set to 700 ul to mix the sample without forming bubbles or vortex at low speed Place the sample on the magnet until the beads have formed a tight pellet and the supernatant is clear Without removing the tube from the magnet carefully aspirate and discard the supernatant without disturbing the pellet by angling the pipette such that the tip is pointed away from the pellet Proceed to Eluting RNA next page Continu
7. Switch Total RNA Cell Kits Lyse sample Bead charge Bind RNA to ChargeSwitch Charge on Magnetic Beads pH lt 6 0 Optional DNase I treatment Wash beads containing RNA Charge on to remove contaminants pH 7 0 Charge off Elute RNA from beads pH gt 8 5 General Information MagnaRack To maximize the RNA yield follow these guidelines for handling RNA when processing your samples e Use RNase AWAY Reagent page viii to remove RNase contamination from surfaces e Maintain an RNAse free working area while processing your samples e Use disposable individually wrapped sterile plastic ware e Use only sterile new pipette tips and microcentrifuge tubes e Always wear latex gloves while handling reagents and RNA samples to prevent RNase contamination from the surface of the skin e Always use proper microbiological aseptic techniques when working with RNA You will need a magnetic rack separator for use with the ChargeSwitch Total RNA Kits We recommend using the MagnaRack to obtain the best results Other magnetic separators may not provide similar magnetic strength or not be compatible with the volumes used in the protocol The MagnaRack available from Invitrogen page viii is a two piece magnetic separation rack for use in protocols with magnetic beads The MagnaRack consists of a magnetic base station and a removable tube rack The tube rack can hold up to 24
8. ce with the most current approved product specifications Current specifications consist of tests for e Bead size charge and binding capacity e Nucleic acid quality and quantity e Buffer turbidity volume and absence of RNases and DNases e Kit packaging and labeling accuracy In addition each kit component must be RNase free and is lot qualified for optimal performance For individual lot test results and more information visit www invitrogen com to download the Certificate of Analysis vii Accessory Products Additional Products RNA Cell Kit The table below lists additional products available from Invitrogen that may be used with the ChargeSwitch total In addition the table lists a selection of ChargeSwitch Kits that are available for the purification of genomic DNA from various sample sources For more information about these or other ChargeSwitch Kits refer to our website at www invitrogen com or contact Technical Service page 20 Product Amount Catalog no MagnaRack Magnetic Rack 1 rack CS15000 RNase AWAY 250 ml 10328 011 0 1 2 Kb RNA Ladder 75 ug 15623 100 0 5 10 Kb RNA Ladder 75 ug 15623 200 UltraPure DEPC treated Water 1L 750023 UltraPure DNase RNase Free Distilled Water 500 ml 10977 015 UltraPure Dithiothreitol 5g 15508 013 Quant iT RNA Assay Kit 1000 assays 033140 ChargeSwitch gDNA 20 ul Blood Kit 96 purifications CS11010 ChargeSwitch gDNA 100 ul B
9. ds have formed a tight pellet and the supernatant is clear Without removing the tube from the magnet carefully aspirate and discard the supernatant without disturbing the pellet by angling the pipette such that the tip is pointed away from the pellet Remove the tube from the magnet Add 250 ul of the prepared DNase I Mix page 11 Resuspend the magnetic beads by pipeting up and down gently 5 times using a 1 ml pipette tip set to 200 ul to mix the sample without forming bubbles or vortex at low speed to avoid any solution from collecting in the cap of the tube Incubate at room temperature for 10 minutes Add 80 ul Binding Buffer B9 to the sample and pipet up and down gently 5 times using a 1 ml pipette tip set to 200 ul to mix the sample without forming bubbles or vortex at low speed to resuspend the magnetic beads Incubate at room temperature for 1 minute Place the sample on the magnet until the beads have formed a tight pellet and the supernatant is clear Without removing the tube from the magnet carefully aspirate and discard the supernatant without disturbing the pellet by angling the pipette such that the tip is pointed away from the pellet Proceed to Washing RNA next page Continued on next page 13 Isolating Total RNA from Cells Continued Washing RNA 14 Optional If you wish to remove DNA from the sample perform DNase I Treatment previous page before Washing RNA If co purification of genomic
10. e prepared Lysis Mix previous page Pipet up and down thoroughly up to 15 times until the cells are lysed and the lysate is no longer viscous Transfer the lysate into a 1 5 ml microcentrifuge tube Incubate at 60 C for 15 minutes After incubation mix the lysate briefly by vortexing Cool the samples for 1 minute on ice Proceed to Binding RNA page 12 Continued on next page Sample Preparation Continued Preparing For best results we recommend using log phase bacterial Bacterial cells OD600 0 6 to 1 2 Lysates 1 Harvest 1 ml overnight bacterial culture by centrifugation Completely remove the growth medium 2 Wash the cells with 1X PBS Centrifuge the cells and completely remove the PBS from the cell pellet Note Incomplete removal of the growth medium will inhibit the lysis and affect the efficiency of the RNA purification 3 Resuspend the cells in 500 ul Lysis Mix page 8 Pipet up and down thoroughly up to 15 times until the pellet is broken up and the lysate is no longer viscous Incubate at 60 C for 15 minutes 5 After incubation vortex the lysate briefly to mix Cool the samples for 1 minute on ice 6 Proceed to Binding RNA page 12 10 Isolating Total RNA from Cells Introduction This procedure is designed for purifying total RNA from lysed mammalian cells page 9 or lysed bacteria page 10 The procedure includes an optional DNase I Treatment for purification of DNA free tota
11. ed on next page Isolating Total RNA from Cells Continued Eluting RNA 1 Remove the tube containing the pelleted magnetic beads from the magnet There should be no supernatant in the tube Add 150 ul Elution Buffer E7 to the tube Resuspend the magnetic beads by pipeting up and down gently 10 times using a pipette tip set to 120 ul to mix the sample or vortex at low speed Note See page 6 for more information on Elution Buffer volume Pre warming the Elution Buffer to 60 70 C may increase the RNA yield Incubate at room temperature for 5 minutes Tip For maximum yield mix the suspension of beads after 2 minutes of incubation by pipetting up and down gently Place the sample on the magnet until the beads have formed a tight pellet and the supernatant is clear Without removing the tube from the magnet carefully transfer the supernatant containing the RNA to a sterile microcentrifuge tube without disturbing the pellet by angling the pipette such that the tip is pointed away from the pellet The eluate volume is 165 yl Discard the used magnetic beads Do not reuse the magnetic beads Store the purified total RNA at 80 C or use the RNA for the desired downstream application 15 Analyzing RNA Yield and Quality RNA Yield Analyze the yield of purified total RNA by checking the UV absorbance at 260 nm or by using the Quant iT RNA Assay Kits UV Absorbance 1 Dilute an aliquot of the total RNA sa
12. et Remove the eluate to a sterile microcentrifuge tube taking care not to disturb the bead pellet DNA Improper DNaseI e Store DNase I at 20 C contamination digestion immediately upon receipt Do not store DNase I at room temperature e Perform DNase I digestion as directed in the protocol RNA is Improper handling Follow the recommendations on page 4 degraded gel of RNA samples to prevent RNase contamination electrophoresis analysis shows a smear 19 Technical Service World Wide Visit the Invitrogen Web site at www invitrogen com for Web e Technical resources including manuals vector maps and sequences application notes MSDSs FAQs formulations citations handbooks etc e Complete technical service contact information e Access to the Invitrogen Online Catalog e Additional product information and special offers Contact Us For more information or technical assistance call write fax or email Additional international offices are listed on our Web page www invitrogen com Corporate Headquarters European Headquarters Invitrogen Corporation Invitrogen Ltd 1600 Faraday Avenue Inchinnan Business Park Carlsbad CA 92008 USA 3 Fountain Drive Tel 1 760 603 7200 Paisley PA4 9RF UK Tel Toll Free 1 800 955 6288 Tel 44 0 141 814 6100 Fax 1 760 602 6500 Tech Fax 44 0 141 814 6117 E mail E mail tech_support invitrogen com eurotech invitrogen com Continued on n
13. ext page 20 Technical Service Continued MSDS Requests Limited Warranty To request an MSDS visit our Web site at www invitrogen com On the home page go to Technical Resources select MSDS and follow instructions on the page Invitrogen is committed to providing our customers with high quality goods and services Our goal is to ensure that every customer is 100 satisfied with our products and our service If you should have any questions or concerns about an Invitrogen product or service contact our Technical Service Representatives Invitrogen warrants that all of its products will perform according to specifications stated on the certificate of analysis The company will replace free of charge any product that does not meet those specifications This warranty limits Invitrogen Corporation s liability only to the cost of the product No warranty is granted for products beyond their listed expiration date No warranty is applicable unless all product components are stored in accordance with instructions Invitrogen reserves the right to select the method s used to analyze a product unless Invitrogen agrees to a specified method in writing prior to acceptance of the order Invitrogen makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Therefore Invitrogen makes no warranty of any kind regarding the contents
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15. invitrogen ChargeSwitch Total RNA Cell Kits For purification of total RNA from cells Catalog nos CS14010 and CS14010S Version C 6 March 2006 25 0821 Table of Contents Table of Contents iii3scisaiiccacnceedeate iasaesetit inca taney tcdaeeiehseieh ew dances ili Kit Contents and Storage i icscccneniods bak nee eee ea ees Vv Product CUUAIIFICAM ON aia cite oi fila xa ch dant saadcescntvea doth aah alteetdadeiWageetnatee vii Accessory Products ix ficicsccicicetenccaley hctuactven cove n ens sdetendecenechenvaiees viii VOI Wrst set cline Cullen E de ce cheat E ETTE 1 Experimental Overview c cccsccceecceeecaeeeeeeeaeeaaeeaaeeaaaeeaeeeaenaes 3 General Information az sssccnechs tania ea raan a asin nedva ye locas cates 4 Sample Preparation cccccceee cece cece eeee tees eeeeeeeeeeeeeeeeeeeeeeneeeees 8 Isolating Total RNA from CellS cceccceeeeeeeeeeeentceeeeeeeeetenees 11 Analyzing RNA Yield and Quality cc eeeeeeeeeeeeeeteeeeeeeeeeeeeee 16 Troubleshooting wiceissseciahicrhsaetetennaetasthset ch cetauuietgehennhakaraeablaaabionet 17 Technical SQnviCe screcteser coder sedccateareucecty toctudes ae cconitenadaedeetela andy 20 Purchaser Nouncatlonizsie Alfie ek ae as eee 22 Kit Contents and Storage Types of Kits Shipping and Storage This manual is supplied with the following products Product Catalog no Number of Purifications ChargeSwitch Total
16. l RNA To obtain high quality total RNA follow the guidelines recommended for handling RNA page 4 Materials You will need the following items Needed e Sample lysate prepared as described in the Sample Preparation section page 8 e MagnaRack page 4 e Sterile 1 5 ml microcentrifuge tubes e Adjustable pipettes and aerosol barrier pipette tips e Water bath or heat block set at 60 70 C Components Supplied with the Kit e ChargeSwitch Magnetic Beads e ChargeSwitch Binding Buffer B9 e DNase I e DNase I Buffer e ChargeSwitch Wash Buffer W13 e ChargeSwitch Wash Buffer W14 e ChargeSwitch Elution Buffer E7 Preparing DNase I Digestion Mix Reagents For optional DNase I digestion page 13 mix for each sample the following items in a microcentrifuge tube e 5ul DNase I enzyme e 250 ul DNase I Buffer Mix well by pipetting Do not vortex For multiple samples prepare a master DNase I Digestion Mix by scaling up the volume of reagents accordingly Continued on next page 11 Isolating Total RNA from Cells Continued Binding RNA Follow the procedure below to bind the RNA to the ChargeSwitch Magnetic Beads 1 Thoroughly vortex the tube containing the ChargeSwitch Magnetic Beads to fully resuspend the beads in the storage buffer Add 100 pl of ChargeSwitch Magnetic Beads to the lysate Add 200 ul Binding Buffer B9 to the samples and mix by pipetting up and down gently 5 times using
17. lood Kit 50 purifications CS11000 ChargeSwitch gDNA 1 ml Serum Kit 50 purifications CS11040 Phosphate Buffered Saline PBS 1X 500 ml 10010 023 viii Overview Introduction The ChargeSwitch Technology The ChargeSwitch Total RNA Cell Kits allow rapid and efficient purification of total RNA from cells After preparing the lysates you can purify the RNA in less than 15 minutes using the ChargeSwitch Technology For more information on the Charge Switch Technology see below The purified total RNA is suitable for use in any downstream application of choice page 2 The ChargeSwitch Technology is a novel magnetic bead based technology that provides a switchable surface charge dependent on the pH of the surrounding buffer to facilitate nucleic acid purification In low pH conditions the ChargeSwitch beads have a positive charge that binds the negatively charged nucleic acid backbone see figure below Proteins and other contaminants are not bound and are simply washed away in aqueous wash buffers To elute nucleic acids the charge on the surface is neutralized by raising the pH to gt 8 5 using a low salt elution buffer see figure below Purified DNA or RNA elutes instantly into this elution buffer and is ready for use in downstream applications oh enh Low pH High pH Continued on next page Overview Continued Advantages System Specifications Downstream Applications U
18. microcentrifuge tubes The tube rack fits onto the magnetic base station in two different positions associating the row of 12 neodinium magnets with a single row of 12 tubes for simple and easy on the magnet and off the magnet sample processing see figure below For more information see www invitrogen com or contact Technical Service page 20 We Continued on next page General Information Continued Handling Follow the recommendations below for best results Magnetic e Beads Do not freeze the ChargeSwitch Magnetic Beads as freezing irreparably damages the property of the beads for nucleic acid purifications Store the beads at room temperature Always keep the beads in solution Do not allow the beads to dry as this renders them non functional Before using the beads resuspend thoroughly in the storage buffer by vortexing before removal from the storage tube During the mixing and washing steps of the ChargeSwitch Magnetic Beads mix beads by pipetting up and down gently as directed in the protocol You can also vortex at low speed to mix beads as directed in the protocol To avoid any solution from collecting into the cap of the tube always vortex at low speed Always use an adjustable pipette set to a specific volume as directed in the protocol for mixing the contents by pipetting up and down gently to avoid forming bubbles During all washing steps with beads add buffer to the tube con
19. mple in 10 mM Tris HCl pH 7 0 Mix well Transfer to a cuvette 1 cm path length Note The RNA must be in a neutral pH buffer to accurately measure the UV absorbance 2 Determine the OD260 of the sample using a spectrophotometer blanked against 10 mM Tris HCl pH 7 0 3 Calculate the amount of total RNA using the formula Total RNA ug OD260 x 40 pg 1 OD260 x 1 ml x dilution factor x total sample volume ml Quant iT RNA Assay Kits The Quant iI RNA Assay Kit page viii provides a rapid sensitive and specific method for RNA quantitation with minimal interference from DNA protein or other common contaminants that affect UV absorbance readings The kit contains a state of the art quantitation reagent pre diluted standards for standard curve and a ready to use buffer The assay is performed in a microtiter plate format and is designed for reading in standard fluorescent microplate readers RNA Quality Typically RNA isolated using the ChargeSwitch Total RNA Kits has an OD260 280 of gt 1 8 when samples are diluted in Tris HCl pH 7 5 indicating that RNA is reasonably clean of proteins and other UV chromophores heme chlorophyl that could interfere with downstream applications or negatively affect the stability of the stored RNA Agarose gel electrophoresis of purified RNA should show discreet 28S 5 kb and 18S 1 9 kb ribosomal RNA bands and the 28S to 18S band ratio to be gt 1 5 16 Trouble
20. ntitation using UV absorbance is performed with 10 mM Tris HCl pH 7 0 page 16 to accurately measure the UV absorbance Magnetic beads not functional e Ensure that the magnetic beads are fully resuspended before use e Do not freeze the magnetic beads e Store the magnetic beads at room temperature e Do not reuse the magnetic beads Insufficient amount of ChargeSwitch Magnetic Beads used for binding Use the recommended amount of magnetic beads for binding If RNA yields are lower you may increase the amount of beads used To compensate also increase the amount of Binding Buffer B9 by 10 ul for every additional 30 ul of beads used Low RNA content Various cells and tissues have different RNA content and the yield is dependent on the cell type For low yielding samples if concentration is too low decrease the amount of beads used to enable a smaller amount of elution volume To compensate also reduce the amount of Binding Buffer B9 by 10 ul for every additional 30 ul of beads used Bubbles formed during mixing step Ensure that the pipette tip is submerged in the solution during mixing 18 Continued on next page Troubleshooting Continued Problem Cause Solution Eluate Magnetic pellet Place the sample on the MagnaRack containing RNA disturbed during on the magnet position page 4 until is discolored elution the beads form a tight pell
21. of any publications or documentation If you discover an error in any of our publications please report it to our Technical Service Representatives Invitrogen assumes no responsibility or liability for any special incidental indirect or consequential loss or damage whatsoever The above limited warranty is sole and exclusive No other warranty is made whether expressed or implied including any warranty of merchantability or fitness for a particular purpose 21 Purchaser Notification Limited Use Label No 5 Invitrogen Technology 22 The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes The buyer may transfer information or materials made through the use of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a not to transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for Commerc
22. se of the ChargeSwitch Total RNA Cell Kits provide the following advantages e Uses a magnetic bead based technology to isolate high quality total RNA including rRNA and the increasingly important small RNAs smaller than 200 nucleotides e Eliminates the use of hazardous chemicals common enzymatic inhibitors centrifugation or vacuum manifolds e Rapid and efficient purification of total RNA from mammalian or bacterial cells in less than 15 minutes following sample preparation and lysis e Minimal genomic DNA contamination of the purified RNA e Reliable performance of the high quality purified total RNA with improved performance in downstream applications compared to silica based RNA purification Starting Material Mammalian Cells up to 1 x 10 cultured cells Bacterial Cells up to 1 ml culture ODs 0 6 1 2 Bead Binding Capacity 40 ug total RNA per mg Bead Size lt lum Bead Concentration 25 mg ml Bead Storage Buffer 1 mM sodium acetate pH 4 5 Elution Volume 150 ul RNA yields depend on the condition and type of cells Total RNA isolated using the ChargeSwitch Total RNA Cell Kits is suitable for Direct Use Use after reverse transcription Northern blotting RT PCR Nuclease protection assays Real time quantitative PCR qPCR Reverse transcription Experimental Overview Experimental The figure below illustrates the basic steps necessary to purify Outline total RNA from cells using the Charge
23. shooting Introduction Refer to the table below to troubleshoot problems that you may encounter when purifying total RNA with the kit Problem Cause Solution Low RNA Incomplete lysis e Decrease the amount of starting yield material used Be sure to add Proteinase K during lysis Increase the incubation time during lysis Ensure that the sample is fully homogenized before proceeding to 60 C incubation Depending on the type of adherent cells it may be necessary to detach the cells using trypsin to ensure recovery of all cells Ensure that you completely remove the growth medium from the cells After removing the growth medium from the cells wash the cells once in 1X PBS Remove the PBS before adding the Lysis Mix to the cells Quality of starting material The yield and quality of RNA isolated depends on the cell type Incorrect elution conditions After adding Elution Buffer E7 to the sample pipet up and down to resuspend the magnetic beads before incubation Increase the mixing steps during elution to 20 40 times or vortex at low speed for an additional 5 10 seconds Pre heat the Elution Buffer at 60 70 C Always use Elution Buffer E7 to elute the total RNA page 6 for best results Continued on next page 17 Troubleshooting Continued Problem Cause Solution Low RNA yield RNA quantitation performed with water Be sure the RNA qua
24. taining beads while the tube is still in the on the magnet position to prevent drying of beads Subsequently remove the tube from the magnet and resuspend the beads as described above To aspirate the supernatant after bead washing place the pipette tip away from the beads by angling the pipette such that the tip is pointed away from the pellet and carefully remove the supernatant without disturbing or removing any beads see figure below lt Pipette tip lt Magnetic pellet Discard beads after use Do not reuse the beads Continued on next page General Information Continued Elution Buffer Elution Buffer ChargeSwitch Elution Buffer E7 10 mM Tris HCl pH 9 0 is provided in the kit for eluting total RNA from the ChargeSwitch Magnetic beads For best results use Elution Buffer E7 to elute the RNA Alternatively Tris Buffer pH 8 5 9 0 in RNase free water is acceptable Note that if the pH of the buffer is lt 8 5 the RNA will not elute Do not use water for elution Elution Buffer Volume The protocol recommends eluting the RNA in 150 ul ChargeSwitch Elution Buffer You may vary the amount of ChargeSwitch Elution Buffer to obtain total RNA in the desired final concentration For best results always use a volume of ChargeSwitch Elution Buffer E7 that is equal or greater than the volume of ChargeSwitch Magnetic Beads used in the protocol If the volume of ChargeSwitch Elution
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