RNA/Protein Purification 96-Well Kit - Protocol
Contents
1. Low protein content in the starting materials Run a 20 uL fraction from the flowthrough after RNA 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 Proteins are degraded Eluted protein solution was not neutralized Add 9 3 uL of Neutralizer 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 Eluted protein was not neutralized quickly enough If eluted proteins are not used immediately degradation will occur We strongly suggest adding Neutralizer in order to lower the pH Related Products Product RNA DNA Protein Purification Kit 23500 RNA Protein Purification Kit 23000 Total RNA Purification Kit 17200 Total RNA Purification 96 Well Kit 24300 RNase Free DNase Kit 25710 1kb RNA Ladder 15003 UltraRanger 1kb DNA Ladder 12100 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 2013 Norgen Biotek Corp P1I37900 6 192. f Pellet the spheroplasts Centrifuge a 96 well block at 650 x g 800 1 000 RPM for 3 minutes or microcentrifuge tubes at 200 x g 2 000 RPM for 3 minutes and remove the supernatant by pipetting Add 250 uL of Lysis Solution and vortex vigorously for at least 10 seconds Note Atthis stage the lysate may be stored at 70 C such that the RNA purification can be performed at a later time Add 100 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 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 40 mg of fungi be used for this procedure in order to prevent clogging of the column 1F Cell Lysate Preparation from Fungi a b Cc Determine the amount of fungi by weighing It is recommended that no more than 40 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 Allow the liquid nitrogen to evaporate without al
3. cells may be processed for most cell lines 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 cells 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 Frozen pellets should be stored for no longer than 2 weeks to ensure that the integrity of the RNA is not compromised Frozen cell pellets should not be thawed prior to beginning the protocol Add the Lysis Solution directly to the frozen cell pellet Step 1 A ii d 1A i Cell Lysate Preparation from Cells Growing in a Monolayer a b C 1A ii Aspirate media and wash cell monolayer with an appropriate amount of PBS Aspirate PBS Add 250 uL of Lysis Solution directly to culture plate Lyse cells by gently tapping culture dish and swirling buffer around plate surface for five minutes Transfer lysate to a microcentrifuge tube Note Atthis stage the lysate may be stored at 70 C such that the RNA purification can be performed at a later time Add 100 uL of isopropanol provided by the user to the lysate Mix by vortexing for 10 seconds Proceed to Step 2 Cell Lysate Preparation from Cells Growing in Suspension and Lifted Cells Transfer cell suspension to an RNase free tube not provided and centrifuge ina microcentrifuge at no more than 200 x g 2 000 RPM
4. 25 gauge needle genomic DNA attached to a syringe 5 10 times in order to shear the present in sample genomic DNA prior to loading onto the column Clogged Well Centri Ensure that the centrifuge remains at room temperature entrituge hroughout the procedure Temperatures below 20 C temperature too throughout the procedure Temperatures below ow 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 it is P important that the procedure be performed quickly This rocedure not performed quickly is especially important for the Cell Lysate Preparation enough Step in the Animal Tissue protocol since the RNA in animal tissues is not protected after harvesting until it is disrupted and homogenized RNA is Degraded Improper storage of For short term storage RNA samples may be stored at the purified RNA 20 C for a few days It is recommended that samples be stored at 70 C for longer term storage Ensure that the optional DNase being used with this kit DNase used may is 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 Ensure that the lysozyme and
5. Elution and pH Adjustment The supplied Protein Elution Buffer consists of 10 mM sodium phosphate pH 12 5 a Add 9 3 uL of Neutralizer to the appropriate wells of a new 96 Well Elution Plate b Stack the 96 Well Filter Plate on top of the 96 Well Elution Plate containing the Neutralizer to complete the vacuum manifold set up c Apply 100 uL of the Protein Elution Buffer to the wells of the 96 Well Filter Plate d Apply vacuum for 2 minutes to elute the proteins 13 Note Ensure the entire Protein Elution Buffer has passed through into the elution plate by inspecting the 96 Well Filter Plate If the entire elution volume has not passed apply vacuum for an additional 2 minutes e Gently agitate the elution plate to mix the eluent with the Neutralizer Section 3 RNA Protein Purification from All Types of Lysate using Centrifugation Note The remaining steps of the procedure for the purification of total RNA and proteins using centrifugation are the same from this point forward for all the different types of lysate A RNA Purification Using Centrifugation 2 Binding RNA to 96 Well Filter Plate a Place the 96 Well Filter Plate on top of a provided 96 Well Collection Plate b Apply the lysate with the isopropanol from Step 1 into each well of the 96 Well Filter Plate Centrifuge the assembly at maximum speed or 3 000 x g 3 000 RPM for 2 minutes Note Depending on the starting material a small quantity of precipitates may
6. Solution has passed through into the elution plate by inspecting the 96 Well Filter Plate If the entire elution volume has not passed apply vacuum for an additional 2 minutes d Retain the 96 Well Filter Plate for Protein Purification 5 Storage of RNA Use the provided adhesive tape to seal the 96 Well Elution Plate The purified RNA samples may be stored at 20 C for a few days It is recommended that samples be placed at 70 C for long term storage 12 B Total Protein Purification from All Cell Types Using Vacuum 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 loaded directly onto an SDS PAGE gel for visual analysis or the proteins can be further purified using the protocol below e Add 93 mg of DL Dithiothreitol DTT not provided to the Protein Loading Dye The Protein Loading Dye 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 Protein Loading Dye should be used instead of regular SDS PAGE Loading Buffer in order to prevent any precipitates from forming Add 1 volume of the Protein Loading Dye to the sample and boil for 2 minutes before loading 6 pH Adjustment of Lysate a Use 100 uL of flowthrough from the RNA Binding Step Step 2c above Note Up to 200 uL of flowthrough may be used However the re
7. appear in the lysate ethanol mix No additional step is required to remove these precipitates prior to application to the wells c Retain the flowthrough for Protein Purification Section 3B by transferring to another 96 well plate not provided or microcentrifuge tubes not provided The flowthough contains the proteins and should be stored on ice or at 20 C until the Protein Purification protocol is carried out Reassemble the the 96 Well Filter Plate and the bottom plate Note Ensure that all of the lysate from each well has passed through into the bottom plate If the entire lysate volume has not passed centrifuge for an additional 2 minutes Optional Step Norgen s RNA Protein Purification 96 Well Kit isolates total RNA with minimal amounts of genomic DNA contamination However an optional On Plate DNA Removal Protocol is provided in Appendix A for maximum removal of residual DNA that may affect sensitive downstream applications This step should be performed at this point in the protocol 3 RNA Wash a Apply 400 uL of Wash Solution to each well of the 96 Well Filter Plate Centrifuge the assembly at maximum speed or 3 000 x g 3 000 RPM for 2 minutes Note Ensure the entire wash solution has passed through into the bottom plate by inspecting the 96 Well Filter Plate If the entire wash volume has not passed centrifuge for an additional 2 minutes Discard the flowthrough Reassemble the 96 Well Filter Plate and the
8. bottom plate Repeat steps 3a and 3b to wash column for a second time Repeat steps 3a and 3b to wash column for a third time Pat the bottom of the 96 Well Filter Plate dry Reassemble the 96 Well Filter Plate and the bottom plate Centrifuge the assembly at maximum speed or 3 000 x g 3 000 2205 14 RPM for 5 minutes in order to completely dry the plate Retain the 96 Well Collection Plate for Protein Purification 4 RNA Elution a Stack the 96 Well Filter Plate on top of the 96 Well Elution Plate b Add 75 uL of Elution Solution to each well of the 96 Well Filter Plate c Centrifuge the assembly at maximum speed or 3 000 x g 3 000 RPM for 2 minutes Note Ensure the entire Elution Solution has passed through into the elution plate by inspecting the 96 Well Filter Plate If the entire elution volume has not passed apply centrifugation for an additional 2 minutes d Retain the 96 Well Filter Plate for Protein Purification 5 Storage of RNA Use the provided adhesive tape to seal the 96 Well Elution Plate 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 B Total Protein Purification from All Cell Types Using Centrifugation 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 loaded directly onto an SDS PAGE gel for visual analysis or the proteins can be
9. for 10 minutes to pellet cells Carefully decant the supernatant to ensure that the pellet is not dislodged Wash the cell pellet with an appropriate amount of PBS Centrifuge at 200 x g 2 000 RPM for another 5 minutes 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 Add 250 uL of Lysis Solution to the pellet Lyse cells by vortexing for 15 seconds Ensure that the entire pellet is completely dissolved before proceeding to the next step Note At this stage the lysate may be stored at 70 C such that the RNA purification can be performed at a later time Add 100 uL of isopropanol provided by the user to the lysate Mix by vortexing for 10 seconds Proceed to Step 2 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 When isolating total RNA from frozen tissues ensure that the tissue does not thaw during weighing or prior to homogenization The optimal amount of non fibrous tissue be used per w
10. further purified using the protocol below e Add 93 mg of DL Dithiothreitol DTT not provided to the Protein Loading Dye The Protein Loading Dye 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 Protein Loading Dye should be used instead of regular SDS PAGE Loading Buffer in order to prevent any precipitates from forming Add 1 volume of the Protein Loading Dye to the sample and boil for 2 minutes before loading 6 pH Adjustment of Lysate a Use 100 uL of flowthrough from the RNA Binding Step Step 2c above Note Up to 200 uL of flowthrough may be used However the recovery efficiency may be decreased when processing a larger volume b Adjust volume to 400 pL with Molecular Biology Grade Water c Add 16 uL of pH Binding Buffer Mix contents well Note Ifthe entire lysate is to be purified repeat step 6a to 6c with the remaining lysate 7 Protein Binding a Apply the pH adjusted protein samples into each well of the 96 Well Filter Plate Centrifuge the assembly at maximum speed or 3 000 x g 3 000 RPM for 2 minutes b Discard the flowthrough Reassemble the 96 Well Filter Plate with the collection plate c Depending on your sample volume repeat steps 7a and 7b until the entire protein sample has been loaded onto the column 8 Column Wash a Apply 400 uL of Protein Wash Buffer to to each
11. later time Add 120 uL of isopropanol provided by the user to each tissue sample Mix by pipetting up and down a few times 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 200 uL of Lysis Solution to the blood Lyse cells by vortexing for 15 seconds Ensure that mixture becomes transparent before proceeding to the next step Note At this stage the lysate may be stored at 70 C such that the RNA purification can be performed at a later time Add 120 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
12. lyticase being used with lyticase used may this kit is RNase free in order to prevent possible not be RNAse free problems with RNA degradation Starting material For starting materials with high RNAase content it is may have a high recommended that B mercaptoethanol be added to the RNase content Lysis Solution RNA is Degraded Frozen tissues or cell pellets were allowed to thaw prior to RNA isolation 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 18 Problem Possible Cause Solution and Explanation RNA does not perform well in downstream applications RNA was not washed three times with the provided Wash Solution Traces of salt from the binding step may remain in the sample if the well is not washed three times with Wash Solution Salt may interfere with downstream applications and thus must be washed from the well Ethanol carryover Ensure that the dry spin under the RNA Wash procedure is performed in order to remove traces of ethanol prior to elution Ethanol is known to interfere with many downstream applications Poor protein recovery Incorrect pH adjustment of sample Ensure that the pH of the starting protein sample is adjusted to pH 3 5 or lower after the pH Binding Buffer has been added and prior to binding to the column If necessary add additional pH Binding Buffer
13. sensitive downstream applications It is recommended that Norgen s RNase Free DNase Kit Product 25710 be used for this step T For every on column reaction to be performed prepare a mix of 10 uL of DNase I and 65 uL of Enzyme Incubation Buffer using Norgen s RNase Free DNase Kit Product 25710 Mix gently by inverting the tube a few times DO NOT VORTEX Note If using an alternative DNase I prepare a working stock of 0 25 Kunitz unit uL RNase free DNase solution according to the manufacturer s instructions A 75 uL aliquot is required for each column to be treated Perform the appropriate Total RNA Purification Procedure for your starting material up to and including Binding RNA to 96 Well Filter Plate Steps 1 and 2 of all protocols For Vacuum Manifold Apply 400 uL of Wash Solution to each well of the 96 Well Filter Plate Tape the plate or any unused wells using sealing tape or a pad provided by the user according to the vacuum manifold manufacturer s recommendations Apply vacuum for 2 minutes For Centrifugation Apply 400 uL of Wash Solution to each well of the 96 Well Filter Plate Centrifuge the assembly at maximum speed or 3 000 x g 3 000 RPM for 2 minutes Discard the flowthrough Reassemble the 96 Well Filter Plate with the vacuum manifold or the bottom plate Apply 75 uL of the RNase free DNase solution prepared in Step 1 to each well of the 96 Well Filter Plate For Vacu
14. well of the 96 Well Filter Plate Centrifuge the assembly at maximum speed or 3 000 x g 3 000 RPM for 2 minutes b Discard the flowthrough and reassemble the spin column with its collection tube 15 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 9 Protein Elution and pH Adjustment The supplied Protein Elution Buffer consists of 10 mM sodium phosphate pH 12 5 290 59 Add 9 3 uL of Neutralizer to the appropriate wells of a new 96 Well Elution Plate Stack the 96 Well Filter Plate on top of the 96 Well Elution Plate containing the Neutralizer Apply 100 uL of the Protein Elution Buffer to the wells of the 96 Well Filter Plate Centrifuge the assembly at maximum speed or 3 000 x g 3 000 RPM for 2 minutes to elute bound proteins Note Ensure the entire Protein Elution Buffer has passed through into the elution plate by inspecting the 96 Well Filter Plate If the entire elution volume has not passed apply centrifugation for an additional 2 minutes Gently agitate the elution plate to mix the eluent with the Neutralizer Appendix A Protocol for Optional On Plate DNA Removal Norgen s RNA Protein Purification 96 Well Kit isolates total RNA with minimal amounts of genomic DNA contamination However an optional protocol is provided below for maximum removal of residual DNA that may affect
15. 3430 Schmon Parkway gt N Thorold ON Canada L2V 4Y6 Phone 866 667 4362 e 905 227 8848 Fax 905 227 1061 BIOTEK si CORPORATION Email techsupport norgenbiotek com RNA Protein Purification 96 Well Kit Product Insert Product 37900 Norgen s RNA Protein Purification 96 Well Kit provides a rapid method for the high throughput 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 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 96 well column chromatography using Norgen s proprietary resin as the separation matrix The purification could be perf
16. Well Collection Plate can be used as the collection waste tray if desired Apply the lysate with the isopropanol from Step 1 into each well of the 96 Well Filter Plate Tape the plate or any unused wells using sealing tape or pads provided by the user according to the vacuum manifold manufacturer s recommendations Apply vacuum for 2 minutes Note Depending on the starting material a small quantity of precipitates may appear in the lysate ethanol mix No additional step is required to remove these precipitates prior to application of the mixture to the wells Turn off vacuum and ventilate the manifold Retain the flowthrough for Protein Purification Section 2B by transferring to another 96 well plate not provided or 11 microcentrifuge tubes not provided The flowthough contains the proteins and should be stored on ice or at 20 C until the Protein Purification protocol is carried out Reassemble the 96 Well Filter Plate and the vacuum manifold Note Ensure that all of the lysate from each well has passed through into the collection waste tray If the entire lysate volume has not passed apply vacuum for an additional 2 minutes Optional Step Norgen s RNA Protein Purification 96 Well Kit isolates total RNA with minimal amounts of genomic DNA contamination However an optional On Plate DNA Removal Protocol is provided in Appendix A for maximum removal of residual DNA that may affect sensitive downstream applications This
17. ation of RNA Lyse cells or tissue using Lysis Solution Bind RNA to plate B Purification of Proteins Flowthrough Proteins Adjust pH Bind Proteins to Plate Wash RNA Wash Elute RNA Elute Proteins RNA Proteins Procedures For Vacuum Manifold All vacuum steps are performed at room temperature The correct pressure can be calculated using the conversions 1 mbar 100 Pa 0 0394 in Hg 0 75 mm Hg 0 0145 psi For Centrifugation All centrifugation steps are performed at room temperature The correct rom can be calculated using the formula RPM RCF 1 118 x 10 r where RCF 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 for preparation of lysate from different types of starting materials Please ensure that the proper protocol is followed for your sample The user then carry out total RNA and total protein purification using either Section 2 with vacuum or Section 3 with centrifugation Section 1 Preparation of Lysate From Various Cell Types Notes Prior to Use for all RNA Protein 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
18. covery efficiency may be decreased when processing a larger volume b Adjust volume to 400 uL with Molecular Biology Grade Water c Add 16 uL of pH Binding Buffer Mix contents well Note Ifthe entire lysate is to be purified repeat step 6a to 6c with the remaining lysate 7 Protein Binding a Apply the pH adjusted protein samples into each well of the 96 Well Filter Plate Tape the plate or any unused wells using sealing tape or pads provided by the user according to the vacuum manifold manufacturer s recommendations Apply vacuum for 2 minutes b Turn off vacuum and ventilate the manifold Discard the flowthrough c Depending on your sample volume repeat steps 7a and 7b until the entire protein sample has been loaded onto the column 8 Column Wash a Apply 400 uL of Protein Wash Buffer to to each well of the 96 Well Filter Plate Tape the plate or any unused wells using sealing tape or pads provided by the user according to the vacuum manifold manufacturer s recommendations Apply vacuum for 2 minutes b Turn off vacuum and ventilate the manifold Discard the flowthrough Reassemble the 96 Well Filter Plate and the vacuum manifold c Turn off vacuum and ventilate the manifold Pat the bottom of the 96 Well Filter Plate dry Reassemble the 96 Well Filter Plate and the vacuum manifold Apply vacuum for an additional 5 minutes in order to completely dry the plate d Turn off vacuum and ventilate the manifold 9 Protein
19. d to the Wash luti Poor RNA Solution supplied Wash Solution prior to use Recovery Low RNA content in cells or tissues used Different tissues and cells have different RNA contents and thus the expected yield of RNA will vary greatly from these different sources Please check literature to determine the expected RNA content of your starting material Cell Culture Cell monolayer was not washed with PBS Ensure that the cell monolayer is washed with the appropriate amount of PBS in order to remove residual media from cells Yeast Lyticase was not added to the Resuspension Buffer Ensure that the appropriate amount of lyticase is added when making the Resuspension Buffer Bacteria and Yeast All traces of media not removed Ensure that all media is removed prior to the addition of the lysis solution through aspiration 17 Problem Possible Cause Solution and Explanation Insufficient solubilization of cells or tissues Ensure that the appropriate amount of lysis buffer was used for the amount of cells or tissue Insufficient Vacuum Ensure that a vacuum pressure of at least 650 mbar or Clogged Well 25 in Hg is developed Maximum number of cells or amount Refer to specifications to determine if amount of starting of tissue exceeds material falls within kit specifications kit specifications High amounts of The lysate may be passed through a
20. ell of the 96 Well Filter Plate is up to 8 mg However for most tissues except tissues with high cell number such as liver and spleen up to 10 mg could be processed For fibrous tissue such as heart a maximum of 2 mg is recommended 1B Cell Lysate Preparation from Animal Tissues a b C d f Excise the tissue sample from the animal Determine the amount of tissue by weighing It is recommended that no more than 10 mg of tissue be used for each well of the 96 Well Filter Plate Transfer the tissue samples to appropriate vessels for the desired disruption method Add 300 uL of Lysis Solution to each tissue sample Note Ensure that frozen tissues do not thaw during weighing or prior to the addition of Lysis Solution For maximum RNA recovery homogenize frozen tissues to fine powder in liquid nitrogen prior to the addition of Lysis Solution Homogenize the tissues using the appropriate cell disruption tool Note Thorough homogenization is required for optimal performance For tissue inputs of lt 8 mg it is not required to perform centrifugation to remove cell debris if the homogenization is complete For tissue inputs larger than 8 mg or if incomplete cell disruption is suspected centrifuge the lysate at maximum speed for 2 minutes in an appropriate centrifuge Transfer the supernatant to a new 96 well microplate Note At this stage the lysate may be stored at 70 C such that the RNA purification can be performed at a
21. f factors including species growth conditions used and developmental stage Kit Components Component Product 37900 2 x 96 samples Lysis Solution 2x 40mL Wash Solution 2x 30 mL Elution Solution 2 x20 mL Protein Wash Buffer 2x 45 mL Protein pH Binding Buffer 2x4mL Protein Elution Buffer 2x20 mL Protein Neutralizer 2x2mL Protein Loading Dye 2x2mL 96 Well Filter Plate Adhesive Tape 4 96 Well Collection Plate 2 96 Well Elution Plate 4 Product Insert 1 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 Protein Loading Dye 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 necessary precautions recommended by the appropriate authorities in the country of use should be taken when working with whole blood The Lysis Solution co
22. in all cases Steps 2 9 e Please ensure that the correct procedure for preparing the lysate from your starting material is followed e Ensure that all solutions are at room temperature prior to use e Prepare a working concentration of the Wash Solution by adding 90 mL of 95 ethanol provided by the user to the supplied bottle containing the concentrated Wash Solution This will give a final volume of 120 mL The label on the bottle has a box that may be checked to indicate that the ethanol has been added e The volumes stated in each procedure for lysate preparation are the volumes required to prepare samples for each well of the 96 well plate e Optional The use of B mercaptoethanol in lysis is highly recommended for most animal tissues particularly those known to have a 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 Lysis Solution required B mercaptoethanol is toxic and should be dispensed in a fume hood Alternatively the lysis solution can be used as provided e It is important to work quickly during this procedure Section 1A Preparation of Lysate From Various Cell Types 1A Lysate Preparation from Cultured Animal Cells Notes Prior to Use e The recommended input is 5 x 10 cells per well of the 96 Well Filter Plate However up to 1 x 10
23. lowing the tissue to thaw Add 300 uL of Lysis Solution 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 At this stage the lysate may be stored at 70 C such that the RNA purification can be performed at a later time Add 100 uL of isopropanol provided by the user to the lysate Mix by vortexing for 10 seconds Proceed to Step 2 1G Lysate Preparation from Plant Notes Prior to Use The maximum recommended input of plant tissue is 40 mg or 4 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 10 1G Cell Lysate Preparation from Plant a Note Transfer lt 40 mg of plant tissue or 4 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 nitr
24. n TE Buffer o For Gram positive bacteria 3 mg mL lysozyme in TE Buffer For Yeast Protocol Resuspension Buffer with Lyticase o 50mM Tris pH 7 5 o 10mMEDTA o 1M Sorbital o 1 unit uL Lyticase For Fungi Protocol Liquid nitrogen Mortar and pestle For Plant Protocol Liquid nitrogen Mortar and pestle 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 microbiological work stations Clean disposable gloves should be worn at all times when handling reagents samples pipettes disposable tubes etc It is 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 96 Well Kit A Purific
25. n i i Bacteria Type y ths S A 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 that 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 b C Pellet yeast by centrifugation of a 96 well block at 3 000 x g 3 000 RPM for 5 mintues or centrifugation of microcentrifuge tubes 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
26. ntains guanidine salts and should be handled with care Guanidine salt forms highly reactive compounds when combined with bleach thus care must be taken to properly dispose of any of these solutions 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 Customer Supplied Reagents and Equipment You must have the following in order to use the RNA Protein Purification 96 Well Kit For All Protocols e For Vacuum Format o Vacuum manifold with vacuum pump capable of generating a minimum pressure of 650 mbar or 25 in Hg such as Whatman UniVac 3 Vacuum to Collect Manifold o Sealing tape or pads e For Centrifuge Format o Centrifuge with rotor for 96 well plate assembly such as Thermo Fisher IEC Centra CL3 series or Beckman GS 15R 95 100 ethanol Isopropanol DL Dithiothreitol DTT B mercaptoethanol optional Molecular Biology Grade Water Deep 1 mL to 2 mL volume 96 Well Plate or microcentrifuge tubes for flowthrough collection and subsequent pH adjustment for protein purification For Animal Cell Protocol e PBS RNase free For Animal Tissue Protocol Liquid nitrogen Mortar and pestle For Bacterial Protocol Lysozyme containing TE Buffer o For Gram negative bacteria 1 mg mL lysozyme i
27. ogen Allow the liquid nitrogen to evaporate without allowing the tissue to thaw Add 300 uL of Lysis Solution 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 At this stage the lysate may be stored at 70 C such that the RNA purification can be performed at a later time Add 100 uL of isopropanol provided by the user to the lysate Mix by vortexing for 10 seconds Proceed to Step 2 The purification of total RNA from the lysate prepared in Section 1 could be performed using either a vacuum manifold or centrifugation For purification using vacuum please follow the procedure outlined in Section 2A For purification using centrifugation please follow the procedure outlined in Section 2B Section 2 RNA Protein Purification from All Types of Lysate using Vacuum Note The remaining steps of the procedure for the purification of total RNA proteins using a vacuum manifold are the same from this point forward for all the different types of lysate A Total RNA Purification Using Vacuum Manifold 2 Binding RNA to 96 Well Filter Plate a b Cc Assemble the 96 Well Filter Plate and the vacuum manifold according to manufacturer s recommendations Note The provided 96
28. ormed on either a vacuum manifold or using centrifugation The process involves first lysing the cells or tissue of interest with the provided Lysis Solution please see the flow chart on page 4 The Lysis Solution 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 96 Well Filter Plate 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 The bound RNA is then washed with the Wash Solution in order to remove any impurities and the purified RNA is eluted with the Elution Solution 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 96 Well Filter Plate provided with the kit After the RNA has been eluted from the 96 Well Filter Plate the flowth
29. recommended that no more than 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 ODsoo 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 centrifugation of a 96 well block at 3 000 x g 3 000 RPM for 5 mintues or centrifugation of microcentrifuge tubes 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 200 uL of Lysis Solution and vortex vigorously for at least 10 seconds Note At this stage the lysate may be stored at 70 C such that the RNA purification can be performed at a later time Add 120 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 Lysozyme Concentratio
30. rough is then pH adjusted and loaded back onto the plate 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 Advantages e Fast and easy processing using either a vacuum manifold or centrifugation e All 96 Well Filter Plate 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 Specifications Kit Specifications ar 50 ug for RNA Binding Capacity Per Well 150 pg for protein Maximum Loading Volume Per Well 500 uL f 3 All sizes including small RNA Size of RNA Purified lt 200 nt Maximum Amount of Starting Material F Animal Cells 1 x 10 cells Animal Tissues 10 mg Blood 100 uL Bacteria 1 x 10 cells Yeast 1 x 10 cells Fungi 40 mg Plant Tissues 40 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 pg protein Liver 5 mg 12 5 ug RNA Liver 5 mg 55 ug protein average yields will vary depending upon a number o
31. step should be performed at this point in the protocol 3 RNA Wash a Apply 400 uL of Wash Solution to each well of the 96 Well Filter Plate Tape the plate or any unused wells using sealing tape or pads provided by the user according to the vacuum manifold manufacturer s recommendations Apply vacuum for 2 minutes Note Ensure the entire wash solution has passed through into the collection waste tray by inspecting the 96 Well Filter Plate If the entire wash volume has not passed apply vacuum for an additional 2 minutes b Turn off vacuum and ventilate the manifold Discard the flowthrough c Reassemble the 96 Well Filter Plate and the vacuum manifold Repeat steps 3a and 3b to wash column for a second time d Reassemble the 96 Well Filter Plate and the vacuum manifold Repeat steps 3a and 3b to wash column for a third time e Pat the bottom of the 96 Well Filter Plate dry Reassemble the 96 Well Filter Plate and the vacuum manifold Apply vacuum for an additional 5 minutes in order to completely dry the plate f Turn off vacuum and ventilate the manifold Retain the 96 Well Collection Plate if used for Protein Purification 4 RNA Elution a Replace the collection waste tray in the vacuum manifold with the provided 96 Well Elution Plate Complete the vacuum manifold assembly with the 96 Well Filter Plate b Add 75 uL of Elution Solution to each well of the plate c Apply vacuum for 2 minutes Note Ensure the entire Elution
32. um Manifold Apply vacuum for 30 seconds For Centrifugation Centrifuge the assembly at maximum speed or 3 000 x g 3 000 RPM for 30 seconds 16 6 After the centrifugation or vacuum in Step 5 pipette the flowthrough that is present in the collection plate back onto the top of the column Note Ensure Step 6 is performed in order to ensure maximum DNase activity and to obtain maximum yields of RNA in particular for small RNA species 7 Incubate the assembly at 25 30 C for 15 minutes 8 Without any further centrifugation proceed directly to RNA Wash Section 2A Step 3c for Vacuum Manifold procedure or Section 2B Step 3c for Centrifugation procedure Troubleshooting Guide Problem Possible Cause Solution and Explanation Incomplete lysis of Ensure that the appropriate amount of Lysis Solution was cells or tissue used for the amount of cells or tissue Do not exceed the recommended amounts of starting Well has become materials The amount of starting material may need to clogged be decreased if the column shows clogging below the recommended levels See also Clogged Well below Teo Was It is recommended that the Elution Solution supplied with need this kit be used for maximum RNA recovery Alcohol was not Ensure that the appropriate amount of isopropanol is added to the lysate added to the lysate before binding to the column Piano was net Ensure that 90 mL of 95 ethanol is added to the adde
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