Protocol (50 prep) - Norgen Biotek Corp.
Contents
1. cells e Cell pellets can be stored at 70 C for later use or used directly in the procedure Determine the number of cells present before freezing e Frozen pellets should be stored for no longer than 2 weeks to ensure that the integrity of the RNA is not compromised e Frozen cell pellets should not be thawed prior to beginning the protocol Add the EMEA GIN directly to the frozen cell pellet Step 1 A ii d 1A i Cell Lysate Preparation from Cells Growing in a Monolayer a Aspirate media and wash cell monolayer with an appropriate amount of PBS Aspirate PBS b Add 350 uL of Buffer SK directly to culture plate c Lyse cells by gently tapping culture dish and swirling buffer around plate surface for five minutes d Transfer lysate to a microcentrifuge tube e Add 150 uL of isopropanol provided by the user to the lysate Mix by vortexing for 10 seconds Proceed to Step 2 Note For input amounts greater than 10 cells it is recommended that the lysate is passed through a 25 gauge needle attached to a syringe 5 10 times at this point in order to reduce the viscosity of the lysate prior to loading onto the column 1A ii Cell Lysate Preparation from Cells Growing in Suspension and Lifted Cells a Transfer cell suspension to an RNase free tube not provided and centrifuge at no more than 200 x g 2 000 RPM for 10 minutes to pellet cells b Carefully decant the supernatant to ensure that the pellet is not dislodged Was2. every 100 uL of lysate Vortex to mix Proceed to Step 2 1G Lysate Preparation from Plant Notes Prior to Use The maximum recommended input of plant tissue is 50 mg or 5 x 10 plant cells Both fresh and frozen plant samples can be used for this protocol Samples should be flash frozen in liquid nitrogen and transferred immediately to a 70 C freezer for long term storage Do not allow frozen tissues to thaw prior to grinding with the mortar and pestle in order to ensure that the integrity of the RNA is not compromised 1G Cell Lysate Preparation from Plant a Transfer lt 50 mg of plant tissue or 5 x 10 plant cells into a mortar that contains an appropriate amount of liquid nitrogen to cover the sample Grind the sample into a fine powder using a pestle in liquid nitrogen Note If stored frozen samples are used do not allow the samples to thaw before transferring to the liquid nitrogen Allow the liquid nitrogen to evaporate without allowing the tissue to thaw Add 600 uL of Butter SK to the tissue sample and continue to grind until the sample has been homogenized Using a pipette transfer the lysate into an RNase free microcentrifuge tube not provided Spin the lysate for 2 minutes to pellet any cell debris Transfer the supernatant to another RNase free microcentrifuge tube Note the volume of the supernatant lysate Add an equal volume of 70 ethanol provided by the user 100 uL of ethanol is added to every 100 uL
3. washed twice with the provided Wash Solution sample if the column is not washed twice with Wash Solution Salt may interfere with downstream applications and thus must be washed from the column Ethanol carryover Ensure that the dry spin under the Column Wash procedure is performed in order to remove traces of ethanol prior to elution Ethanol is known to interfere with many downstream applications 16 Problem Possible Cause Solution and Explanation Incorrect pH adjustment of sample Poor protein Ensure that the pH of the starting protein sample is adjusted to pH 3 5 or lower after the EIOM ITG has been added and prior to binding to the column If necessary add additional IMACE G recovery Low protein content in the starting materials Run a 20 uL fraction from the flowthrough after Nucleic Acid binding on a SDS PAGE gel to estimate the amount of protein present in the sample In addition use the entire flowthrough in protein purification procedure Eluted protein solution was not Add 9 3 uL of age Ce GTE to each 100 uL of eluted protein in order to adjust the pH to neutral Some proteins are sensitive to high pH such as the elution buffer at pH 12 5 neutralized Proteins are degraded Eluted protein was not neutralized quickly enough If eluted proteins are not used immediately degradation will occur We strongly suggest adding in order to lower the
4. 10 bacterial cells be used in this procedure Bacterial growth can be measured using a spectrophotometer As a general rule an E coli culture containing 1 x 10 cells mL has an ODs of 1 0 For RNA isolation bacteria should be harvested in log phase growth Bacterial pellets can be stored at 70 C for later use or used directly in this procedure Frozen bacterial pellets should not be thawed prior to beginning the protocol Add the Lysozyme containing TE Buffer directly to the frozen bacterial pellet Step 1Dc 1D Cell Lysate Preparation from Bacteria a b C Pellet bacteria by centrifuging at 14 000 x g 14 000 RPM for 1 minute Decant supernatant and carefully remove any remaining media by aspiration Resuspend the bacteria thoroughly in 100 uL of the appropriate lysozyme containing TE Buffer see Table 1 by vortexing Incubate at room temperature for the time indicated in Table 1 Add 300 uL of Buffer SK and vortex vigorously for at least 10 seconds Add 150 uL of isopropanol provided by the user to the lysate Mix by vortexing for 10 seconds Proceed to Step 2 Table 1 Incubation Time for Different Bacterial Strains z Lysozyme Concentration i i Bacteria Type y A TE Bular Incubation Time Gram negative 1 mg mL 5 min Gram positive 3 mg mL 10 min 1E Lysate Preparation from Yeast Notes Prior to Use Prepare the appropriate amount of Lyticase containing Resuspension Buffer considering t
5. Buffer C Protein Neutralizer Protein Binding Protein Elution Protein Wash Protein Eluent Neutralization SDS PAGE Gel Loading Protein Loading Dye Mini Spin Columns 50 Collection Tubes 150 Product Insert 1 Advantages e Fast and easy processing using rapid spin column format e All columns for RNA purification and protein purification provided e Sequentially isolate nucleic acids and proteins from a single lysate no need to split the lysate Isolate total RNA from large rRNA down to microRNA miRNA No phenol or chloroform extractions Isolate high quality total RNA High yields of isolated proteins Storage Conditions and Product Stability All solutions should be kept tightly sealed and stored at room temperature These reagents should remain stable for at least 1 year in their unopened containers The pige tial Mok Co Tate Pye should be stored at 20 C after the addition of DL Dithiothreitol DTT Precautions and Disclaimers This kit is designed for research purposes only It is not intended for human or diagnostic use Ensure that a suitable lab coat disposable gloves and protective goggles are worn when working with chemicals For more information please consult the appropriate Material Safety Data Sheets MSDSs These are available as convenient PDF files online at www norgenbiotek com Blood of all human and animal subjects is considered potentially infectious All
6. for 10 minutes at 14 000 x g 12 000 RPM Discard the supernatant and allow the pellet to air dry Note At this point the pellet can be washed with 100 uL of ice cold ethanol and again air dried d Resuspend the pellet in the buffer of your choice that is suited to your downstream application 14 Troubleshooting Guide Problem Possible Cause Solution and Explanation Incomplete lysis of Ensure that the appropriate amount of EMIAAJYX was cells or tissue used for the amount of cells or tissue Do not exceed the recommended amounts of starting Gann has materials The amount of starting material may need to become clogged be decreased if the column shows clogging below the 99 recommended levels See also Clogged Column below eae Was It is recommended that the AMIE M supplied sed with this kit be used for maximum RNA recovery Ensure that the appropriate amount of isopropanol or n a e ethanol is added to the lysate before binding to the y column Seale ae Ensure that 90 mL of 96 100 ethanol is added to the Poor RNA Solution supplied Wash Solution prior to use Recovery Different tissues and cells have different RNA contents Low RNA content and thus the expected yield of RNA will vary greatly from in cells or tissues these different sources Please check literature to used determine the expected RNA content of your starting material Cell Culture Cell Ensure that the cell monol
7. gt k 3430 Schmon Parkway Thorold ON Canada L2V 4Y6 i Phone 866 667 4362 e 905 227 8848 Fax 905 227 1061 BIOTEK amp CORPORATION Email techsupport norgenbiotek com RNA Protein Purification Kit Product Insert Product 24100 Norgen s RNA Protein Purification Kit provides a rapid method for the isolation and purification of total RNA and proteins simultaneously from a single sample of cultured animal cells small tissue samples blood bacteria yeast fungi or plants The total RNA and proteins are both column purified in under 25 minutes using a single column It is often necessary to isolate total RNA and proteins from a single sample such as for studies of gene expression including gene silencing experiments mRNA knockdowns or experiments correlating RNA and protein expression levels Traditionally the RNA and proteins would be isolated from different aliquots of the same sample however this novel technology will allow for their simultaneous isolation from the same sample This will not only save time but will also be of a great benefit when isolating RNA and proteins from precious difficult to obtain or very small samples Furthermore gene expression analysis will be more reliable since the RNA and proteins are derived from the same sample therefore eliminating inconsistent results Norgen s Purification Technology RNA Purification Purification is based on spin column chromatography using Norgen s proprietary res
8. necessary precautions recommended by the appropriate authorities in the country of use should be taken when working with whole blood Customer Supplied Reagents and Equipment You must have the following in order to use the RNA Protein Purification Kit For All Protocols e Benchtop microcentrifuge B mercaptoethanol Optional 96 100 ethanol Isopropanol DL Dithiothreitol RNase free DNase Optional Molecular biology grade water Milli Q water 1 7 mL microcentrifuge tubes For Animal Cell Protocol e PBS RNase free For Animal Tissue Protocol e Liquid nitrogen e Mortar and pestle e 70 ethanol For Bacterial Protocol Lysozyme containing TE Buffer o For Gram negative bacteria 1 mg mL lysozyme in TE Buffer o For Gram positive bacteria 3 mg mL lysozyme in TE Buffer For Yeast Protocol Resuspension Buffer with Lyticase 50 mM Tris pH 7 5 o 10mMEDTA o 1M Sorbital o 1 unit uL Lyticase O For Fungi Protocol Liquid nitrogen Mortar and pestle 70 ethanol For Plant Protocol Liquid nitrogen Mortar and pestle 70 ethanol Working with RNA RNases are very stable and robust enzymes that degrade RNA Autoclaving solutions and glassware is not always sufficient to actively remove these enzymes The first step when preparing to work with RNA is to create an RNase free environment The following precautions are recommended as your best defense against these enzymes The RNA area should be located away from mic
9. of lysate Vortex to mix Proceed to Step 2 Section 1B Total RNA Purification from All Types of Lysate Note The remaining steps of the procedure for the purification of total RNA are the same from this point forward for all the different types of lysate 2 Binding RNA to Column a Assemble a column with one of the provided collection tubes b Apply up to 600 uL of the lysate with the alcohol onto the column and centrifuge for 1 e minute at 2 3 500 x g 6 000 RPM Note Ensure the entire lysate volume has passed through into the collection tube by inspecting the column If the entire lysate volume has not passed spin for an additional minute at 14 000 x g 14 000 RPM Retain the flowthrough for Protein Purification Section 2 The flowthough contains the proteins and should be stored on ice or at 20 C until the Protein Purification protocol is carried out Depending on your lysate volume repeat steps 2b and 2c if necessary The flowthroughs should be combined and retained in the same microcentrifuge tube Reassemble the spin column with a new collection tube 11 3 DNase Treatment Optional This optional step is carried out if genomic DNA free RNA is required It is recommended that Norgen s RNase Free DNase Kit Product 25710 be used for this step a Apply 400 uL of Wash Solution to the column and centrifuge for 2 minutes Discard the flowthrough Note Ensure the entire wash solution has passed throug
10. pH Related Products Product RNA DNA Protein Purification Kit 24000 RNase Free DNase Kit 25710 1kb RNA Ladder 15003 UltraRanger 1kb DNA Ladder 12100 Technical Support Contact our Technical Support Team between the hours of 8 30 and 5 30 Eastern Standard Time at 905 227 8848 or Toll Free at 1 866 667 4362 Technical support can also be obtained from our website www norgenbiotek com or through email at techsupport norgenbiotek com 3430 Schmon Parkway Thorold ON Canada L2V 4Y6 Phone 905 227 8848 Fax 905 227 1061 Toll Free in North America 1 866 667 4362 2014 Norgen Biotek Corp P124100 5 M14 17
11. CF required gravitational acceleration relative centrifugal force in units of g r radius of the rotor in cm and RPM the number of revolutions per minute required to achieve the necessary g force NOTE This procedure is written in two steps Section 1 contains the protocols to isolate total RNA from different types of starting materials Please ensure that the proper protocol is followed for your sample Section 2 contains the protocol to isolate total proteins from the sample and the same protocol will apply to all the different starting materials Section 1 Procedures to Purify Total RNA from Various Cell Types Notes Prior to Use for all Total RNA Purification Procedures e The steps for preparing the lysate are different depending on the starting material Step 1 However the subsequent steps are the same in all cases Steps 2 6 e Please ensure that the correct procedure for preparing the lysate from your starting material is followed e All centrifugation steps are carried out in a benchtop microcentrifuge at 14 000 x g 14 000 RPM except where noted All centrifugation steps are performed at room temperature e A variable speed centrifuge should be used for maximum kit performance If a variable speed centrifuge is not available a fixed speed centrifuge can be used however reduced yields may be observed e Ensure that all solutions are at room temperature prior to use e Prepare a working concentration of the by
12. adding 90 mL of 96 100 ethanol provided by the user to the supplied bottle containing the concentrated TARJ This will give a final volume of 128 mL The label on the bottle has a box that may be checked to indicate that the ethanol has been added e Add 93 mg of DL Dithiothreitol DTT not provided to the Cuma lem ts The should be stored at 20 C after the addition of DTT The label on the bottle has a box that may be checked to indicate that DTT has been added e Optional The use of B mercaptoethanol in lysis is highly recommended for most animal tissues particularly those known to have high RNAse content ex pancreas as well as for most plant tissues It is also recommended for users who wish to isolate RNA for sensitive downstream applications Add 10 uL of B mercaptoethanol provided by the user to each 1 mL of EMAX required B mercaptoethanol is toxic and should be dispensed in a fume hood Alternatively the EMIAAJX can be used as provided e It is important to work quickly when purifying RNA Section 1A Preparation of Lysate From Various Cell Types 1A Lysate Preparation from Cultured Animal Cells Notes Prior to Use e For optimal results it is recommended that 1 x 10 cells be used for the input However inputs of up to 3 x 10 cells may be used e Ahemocytometer can be used in conjunction with a microscope to count the number of cells As a general guideline a confluent 3 5 cm plate of HeLa cells will contain 10
13. ayer is washed with the monolayer was not appropriate amount of PBS in order to remove residual washed with PBS media from cells Yeast Lyticase was not added to Ensure that the appropriate amount of lyticase is added the Resuspension when making the Resuspension Buffer Buffer P e Ensure that all media is removed prior to the addition of not removed the through aspiration r of Ensure that the appropriate amount of lysis buffer was cells ortissues used for the amount of cells or tissue Clogged Column Maximum number of cells or amount of tissue exceeds kit specifications Refer to specifications to determine if amount of starting material falls within kit specifications 15 Problem Possible Cause Solution and Explanation High amounts of genomic DNA present in sample The lysate may be passed through a 25 gauge needle attached to a syringe 5 10 times in order to shear the genomic DNA prior to loading onto the column Clogged Column Centrifuge Ensure that the centrifuge remains at room temperature g throughout the procedure Temperatures below 20 C temperature too As low may cause precipitates to form that can cause the columns to clog RNases may be introduced during the use of the kit RNase Ensure proper procedures are followed when working contamination with RNA Please refer to Working with RNA at the beginning of this user guide In order to maintain the integrity of the RNA
14. column with its collection tube e Spin the column for 2 minutes in order to thoroughly dry the resin Discard the collection tube 5 RNA Elution a Place the column into a fresh 1 7 mL microcentrifuge tube provided by the user b Add 50 uL of to the column c Centrifuge for 2 minutes at 200 x g 2 000 RPM followed by a1 minute spin at 14 000 x g 14 000 RPM Note the volume eluted from the column If the entire volume has not been eluted spin the column at 14 000 x g 14 000 RPM for 1 additional minute Note For maximum nucleic acid recovery it is recommended that a second elution be performed into a separate microcentrifuge tube Repeat Steps 5b and 5c 12 d Retain the column for Protein Purification Proceed to Section 2 for Protein Purification 6 Storage of RNA The purified RNA sample may be stored at 20 C for a few days It is recommended that samples be placed at 70 C for long term storage Section 2 Procedure to Isolate Total Proteins from All Cell Types A Total Protein Purification from All Cell Types Notes Prior to Use e Atthis point the proteins that are present in the flowthrough from the RNA Binding Step Step 2 above can be processed by one of the following three options o Direct running on an SDS PAGE gel with the provided loading dye for visual analysis o Column purification recommended o Acetone precipitation e Add 93 mg of DL Dithiothreito DTT not provided to the SAONE DIA T
15. commended input of tissue varies depending on the type of tissue being used Please refer to Table 1 below as a guideline for maximum tissue input amounts If your tissue of interest is not included in the table below we recommend starting with an input of no more than 10 mg Table 1 Recommended Maximum Input Amounts of Different Tissues Tissue Maximum Input Amount Brain 25 mg Heart 5 mg Kidney 10 mg Liver 10 mg Lung 10 mg Spleen 10 mg 1B Cell Lysate Preparation from Animal Tissues a b Excise the tissue sample from the animal Determine the amount of tissue by weighing Please refer to Table 1 for the recommended maximum input amounts of different tissues For tissues not included in the table we recommend starting with an input of no more than 10 mg Transfer the tissue into a mortar that contains an appropriate amount of liquid nitrogen to cover the sample Grind the tissue thoroughly using a pestle Allow the liquid nitrogen to evaporate without allowing the tissue to thaw Add 600 uL of Buffer SK to the tissue sample and continue to grind until the sample has been homogenized Homogenize by passing the lysate 5 10 times through a 25 gauge needle attached to a syringe Using a pipette transfer the lysate into an RNase free microcentrifuge tube not provided Spin lysate for 2 minutes to pellet any cell debris Transfer the supernatant to another RNase free microcentrifuge tube not provide
16. d Note the volume of the supernatant lysate Add an equal volume of 70 ethanol provided by the user to the lysate 100 uL of ethanol is added to every 100 uL of lysate Mix by vortexing for 10 seconds Proceed to Step 2 1C Lysate Preparation from Blood Notes Prior to Use Blood of all human and animal subjects is considered potentially infectious All necessary precautions recommended by the appropriate authorities in the country of use should be taken when working with whole blood It is recommended that no more than 100 uL of blood be used in order to prevent clogging of the column We recommend the use of this kit to isolate RNA from non coagulating fresh blood using EDTA as the anti coagulant 1C Cell Lysate Preparation from Blood a b Transfer up to 100 uL of non coagulating blood to an RNase free microcentrifuge tube not provided Add 350 uL of Buffer SK to the blood Lyse cells by vortexing for 15 seconds Ensure that mixture becomes transparent before proceeding to the next step Add 150 uL of isopropanol provided by the user to the lysate Mix by vortexing for 10 seconds Proceed to Step 2 1D Lysate Preparation from Bacteria Notes Prior to Use Prepare the appropriate lysozyme containing TE Buffer as indicated in Table 1 This solution should be prepared with sterile RNAse free TE Buffer and kept on ice until needed These reagents are to be provided by the user It is recommended that no more than
17. h into the collection tube by inspecting the column If the entire wash volume has not passed spin for an additional minute b Apply 100 uL of HAAA oF ilel ae Tica mix containing of 15 uL of Norgen s RNase free DNase Product 25710 to the column and centrifuge at 14 000 x g 14 000 RPM for 1 minute If using an alternative DNase apply 100 uL of MATEG containing 25 units of DNase to the column and centrifuge for 1 minute Note Ensure that the entire volume of DNase mix passes through the column If needed spin at 14 000 x g for an additional minute c After the centrifugation in Step b pipette the flowthrough that is present in the collection tube back onto the top of the column Note Ensure that Step c is performed in order to ensure maximum DNase activity and to obtain maximum yields of RNA in particular for small RNA species d Incubate at room temperature for 15 minutes e Proceed to Step 4c without further centrifugation 4 Column Wash a Apply 400 uL of WEGE Mite Wi to the column and centrifuge for 1 minute Note Ensure the entire wash solution has passed through into the collection tube by inspecting the column If the entire wash volume has not passed spin for an additional minute b Discard the flowthrough and reassemble the column with the collection tube c Wash column a second time by adding another 400 uL of and centrifuge for 1 minute d Discard the flowthrough and reassemble the spin
18. h the cell pellet with an appropriate amount of PBS Centrifuge at 200 x g 2 000 RPM for another 5 minutes c Carefully decant the supernatant A few uL of PBS may be left behind with the pellet in order to ensure that the pellet is not dislodged d Add 350 uL of Buffer SK to the pellet Lyse cells by vortexing for 15 seconds Ensure that the entire pellet is completely dissolved before proceeding to the next step e Add 150 uL of isopropanol provided by the user to the lysate Mix by vortexing for 10 seconds Proceed to Step 2 Note For input amounts greater than 10 cells it is recommended that the lysate is passed through a 25 gauge needle attached to a syringe 5 10 times at this point in order to reduce the viscosity of the lysate prior to loading onto the column 1B Lysate Preparation from Animal Tissues Notes Prior to Use RNA in animal tissues is not protected after harvesting until it is disrupted and homogenized Thus it is important that the procedure is carried out as quickly as possible particularly the Cell Lysate Preparation step Fresh or frozen tissues may be used for the procedure Tissues should be flash frozen in liquid nitrogen and transferred immediately to a 70 C freezer for long term storage Tissues may be stored at 70 C for several months Do not allow frozen tissues to thaw prior to grinding with the mortar and pestle in order to ensure that the integrity of the RNA is not compromised The maximum re
19. hat 500 uL of buffer is required for each preparation The Resuspension Buffer should have the following composition 50 mM Tris pH 7 5 10 mM EDTA 1M Sorbital 0 1 B mercaptoethanol and 1 unit uL Lyticase This solution should be prepared with sterile RNAse free reagents and kept on ice until needed These reagents are to be provided by the user It is recommended that no more than 10 yeast cells or 1 mL of culture be used for this procedure For RNA isolation yeast should be harvested in log phase growth Yeast can be stored at 70 C for later use or used directly in this procedure Frozen yeast pellets should not be thawed prior to beginning the protocol Add the Lyticase containing Resuspension Buffer directly to the frozen yeast pellet Step 1Ec 1E Cell Lysate Preparation from Yeast a Pellet yeast by centrifuging at 14 000 x g 14 000 RPM for 1 minute Decant supernatant and carefully remove any remaining media by aspiration Resuspend the yeast thoroughly in 500 uL of Lyticase containing Resuspension Buffer by vortexing Incubate at 37 C for 10 minutes Pellet the spheroplasts at 200 x g 2 000 RPM for 3 minutes Decant supernatant Add 350 uL of Buffer SK and vortex vigorously for at least 10 seconds Add 150 uL of isopropanol provided by the user to the lysate Mix by vortexing for 10 seconds Proceed to Step 2 1F Lysate Preparation from Fungi Notes Prior to Use Fresh or frozen fungi may be used for
20. he should be stored at 20 C after the addition of DTT The label on the bottle has a box that may be checked to indicate that DTT has been added e For direct running on a gel the provided should be used instead of regular SDS PAGE Loading Buffer in order to prevent any precipitates from forming Add 1 volume of the to the sample and boil for 2 minutes before loading e Column purification of the proteins is recommended For column purification please follow steps 7 to 10 below e For acetone precipitation please refer to the supplementary protocol provided in the Appendix A below 7 pH Adjustment of Lysate a Use 100 uL of flowthrough from the RNA Binding Step Step 2c above Note Up to 300 uL of flowthrough may be used However the recovery efficiency may be decreased when processing a larger volume b Adjust volume to 575 uL with Molecular Biology Grade Water c Add 24 uL of Eiei Mix contents well Note Ifthe entire lysate is to be purified repeat step 7a to 7c with the remaining lysate 8 Protein Binding a Apply up to 600 uL of the pH adjusted protein sample onto the column and centrifuge for 2 minutes at 5 200 x g 8 000 RPM Inspect the column to ensure that the entire 13 sample has passed through into the collection tube If necessary spin for an additional 3 minutes b Discard the flowthrough Reassemble the spin column with its collection tube Note You can save the flowthrough in a fresh tube for assess
21. in as the separation matrix The process involves first lysing the cells or tissue of interest with the provided Buffer SK please see the flow chart on page 4 The Buffer SK contains detergents as well as large amounts of a chaotropic denaturant that will rapidly inactivate RNases and proteases that are present Alcohol is then added to the lysate and the solution is loaded onto a spin column Norgen s resin binds nucleic acids in a manner that depends on ionic concentrations thus only the RNA will bind to the column while the proteins are removed in the flowthrough Next an optional step can be carried out in which the genomic DNA can be digested allowing for a more pure RNA sample to be isolated The bound RNA is then washed twice with the provided Wash Solution A in order to remove any impurities and the purified RNA is eluted with the Elution Solution A The kit purifies all sizes of RNA from large mRNA and ribosomal RNA down to microRNA miRNA and small interfering RNA siRNA The purified RNA is of the highest integrity and can be used in a number of downstream applications including real time PCR reverse transcription PCR Northern blotting RNase protection and primer extension and expression array assays Protein Purification The proteins that are present from the initial flowthrough can now be loaded directly onto an SDS PAGE gel for visual analysis Alternatively the protein samples can be further purified using the spin colu
22. ing your protein s binding efficiency c Depending on your sample volume repeat steps 8a and 8b until the entire protein sample has been loaded onto the column 9 Column Wash a Apply 500 uL of to the column and centrifuge for 2 minutes at 5 200 x g 8 000 RPM b Discard the flowthrough and reassemble the spin column with its collection tube c Inspect the column to ensure that the liquid has passed through into the collection tube There should be no liquid in the column If necessary spin for an additional minute to dry 10 Protein Elution and pH Adjustment The supplied AMIM GAA consists of 10 mM sodium phosphate pH 12 5 a Add 9 3 uL of ei Gime telir ey to a fresh 1 7 mL microcentrifuge tube provided by the user b Transfer the spin column from the Column Wash procedure into the Elution Tube c Apply 100 uL of the Sltiieyam iietale to the column and centrifuge for 2 minutes at 5 200 x g 8 000 RPM to elute bound proteins Note Approximately 95 of bound protein is recovered in the first elution If desired a second elution using 50 uL of AMTES may be carried out This should be collected into a different tube to which 4 6 uL of ge CT NE El is pre added to prevent dilution of the first elution Appendix A Acetone Precipitation Procedure for Proteins a Add 4 volumes of ice cold acetone to the flowthrough from the RNA Binding Step Step 2 b Incubate for 15 minutes on ice or at 20 C c Centrifuge
23. it is Procedur n t important that the procedure be performed quickly This erl rm d duicki is especially important for the Cell Lysate Preparation ee h q y Step in the Animal Tissue protocol since the RNA in g animal tissues is not protected after harvesting until it is disrupted and homogenized improper storage of For short term storage RNA samples may be stored at ha icd A 20 C for a few days It is recommended that samples P be stored at 70 C for longer term storage RNA is Ensure that the DNase being used with this kit is Degraded DNase used may RNase free in order to prevent possible problems with not be RNase free RNA degradation Norgen s RNase Free DNase Kit Cat 25710 is recommended for this step Lysozyme or lyticase used may not be RNAse free Ensure that the lysozyme and lyticase being used with this kit is RNase free in order to prevent possible problems with RNA degradation Starting material may have a high RNase content For starting materials with high RNAase content it is recommended that B mercaptoethanol be added to the Frozen tissues or cell pellets were allowed to thaw Do not allow frozen tissues to thaw prior to grinding with the mortar and pestle in order to ensure that the integrity RNA does not perform well in downstream applications prior to RNA of the RNA is not compromised isolation RNA was not Traces of salt from the binding step may remain in the
24. mns provided with the kit After the RNA has been eluted from the column the flowthrough is then pH adjusted and loaded back onto the column in order to bind the proteins that are present The bound proteins are washed with the provided wash buffer and are then eluted such that they can be used in downstream applications The purified proteins can be used in a number of downstream applications including SDS PAGE analysis or Western blots Specifications Kit Specifications Maximum Column Binding Capacity 50 ug for RNA 200 ug for protein Maximum Column Loading Volume 650 uL Size of RNA Purified All sizes including small RNA lt 200 nt Maximum Amount of Starting Material Animal Cells 3 x 10 cells Animal Tissues 10 mg for most tissues Blood 100 uL Bacteria 1 x 10 cells Yeast 1 x 10 cells Fungi 50 mg Plant Tissues 50 mg Time to Complete 10 Purifications 25 minutes Average Yields HeLa Cells 1 x 10 cells 15 pg RNA HeLa Cells 1 x 10 cells 150 ug protein Liver 5 mg 12 5 ug RNA Liver 5 mg 55 ug protein average yields will vary depending upon a number of factors including species growth conditions used and developmental stage Kit Components Used For RNA Lysis RNA Wash RNA Elution Component Buffer SK Wash Solution A Elution Solution A Product 24100 50 samples Enzyme Incubation Buffer A DNA Removal Wash Solution C Binding Buffer A Elution
25. robiological work stations Clean disposable gloves should be worn at all times when handling reagents samples pipettes disposable tubes etc Itis recommended that gloves are changed frequently to avoid contamination There should be designated solutions tips tubes lab coats pipettes etc for RNA only All RNA solutions should be prepared using at least 0 05 DEPC treated autoclaved water or molecular biology grade nuclease free water Clean all surfaces with commercially available RNase decontamination solutions When working with purified RNA samples ensure that they remain on ice during downstream applications Flow Chart Procedure for Purifying Total RNA and Proteins using Norgen s RNA Protein Purification Kit A Purification of RNA Lyse cells or tissue using Buffer SK Bind RNA to column ea poe B Purification of Proteins SPIN 3 f Flowthrough 5 gt Bind Proteins to f Proteins 2 Column i Wash RNA Adjust pH n u SPIN 3 SPIN 2 f g Wash Elute RNA ii ul SPIN 3 SPIN 3 f Elute Proteins ul SPIN 3 RNA Proteins Procedures All centrifugation steps are carried out in a benchtop microcentrifuge Various speeds are required for different steps so please check your microcentrifuge specifications to ensure that it is capable of the proper speeds All centrifugation steps are performed at room temperature The correct rpm can be calculated using the formula RPM RCF 1 118 x 105 r where R
26. this procedure Fungal tissue should be flash frozen in liquid nitrogen and transferred immediately to a 70 C freezer for long term storage Fungi may be stored at 70 C for several months Do not allow frozen tissues to thaw prior to grinding with the mortar and pestle in order to ensure that the integrity of the RNA is not compromised It is recommended that no more than 50 mg of fungi be used for this procedure in order to prevent clogging of the column 1F Cell Lysate Preparation from Fungi a b Determine the amount of fungi by weighing It is recommended that no more than 50 mg of fungi be used for the protocol Transfer the fungus into a mortar that contains an appropriate amount of liquid nitrogen to cover the sample Grind the fungus thoroughly using a pestle Note At this stage the ground fungus may be stored at 70 C such that the RNA purification can be performed at a later time Allow the liquid nitrogen to evaporate without allowing the tissue to thaw Add 600 uL of Buffer SK to the tissue sample and continue to grind until the sample has been homogenized Using a pipette transfer the lysate into an RNase free microcentrifuge tube not provided Spin the lysate for 2 minutes to pellet any cell debris Transfer the supernatant to another RNase free microcentrifuge tube Note the volume of the supernatant lysate 10 g Add an equal volume of 70 ethanol provided by the user 100 uL of ethanol is added to
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