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1. d Depending on your lysate volume repeat Step 3b and 3c as necessary 4 Column Wash a Apply 400 uL of Wash Solution A 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 spin column with its collection tube Repeat steps 4a and 4b to wash column a second time d Wash column a third time by adding another 400 uL of Wash Solution A and centrifuging for 1 minute e Discard the flowthrough and reassemble the spin column with its collection tube Spin the column for 2 minutes in order to thoroughly dry the resin Discard the collection tube 9 5 RNA Elution a Place the column into a fresh 1 7 mL Elution tube provided with the kit b Add 50 uL of Elution Solution A to the column c Centrifuge for 2 minutes at 200 x g 2 000 RPM followed by 1 minute at 14 000 x g 14 000 RPM Note the volume eluted from the column If the entire 50 uL has not been eluted spin the column at 14 000 x g 14 000 RPM for 1 additional minute Note For maximum RNA recovery it is recommended that a second elution be performed into a separate microcentrifuge tube Repeat Steps 5b and 5c 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 C2. b C 1A ii Aspirate media and wash cell monolayer with an appropriate amount of PBS Aspirate PBS Add 350 uL of Buffer RL 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 Proceed to Step 2 page 11 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 shear the genomic DNA prior to loading onto the column Cell Lysate Preparation from Cells Growing in Suspension and Lifted Cells 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 Carefully decant the supernatant A few uL of media may be left behind with the pellet in order to ensure that the pellet is not dislodged Add 350 uL of Buffer RL to the pellet Lyse cells by vortexing for 15 seconds Ensure that the entire pellet is completely dissolved before proceeding to the next step Proceed to Step 2 page 11 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 shear the genomic DNA prior to loading onto the column 1B Lysate Preparation from Animal Tissues Notes Prior to Use Norgen s Total RNA Purification
3. then added to the flowthrough of the DNA removal step and the solution is loaded onto an RNA Purification Column Norgen s resin binds RNA in a manner that depends on ionic concentrations Thus only the RNA will bind to the column while the contaminating proteins will be removed in the flowthrough or retained on the top of the resin The bound RNA is then washed with the provided Wash Solution A in order to remove any remaining impurities and the purified total RNA is eluted with the Elution Solution A The purified RNA is of the highest integrity and can be used in a number of downstream applications Specifications Kit Specifications Maximum Column Binding Capacity 50 ug Maximum Column Loading Volume 650 uL f as All sizes including small RNA Size of RNA Purified lt 200 nt Maximum Amount of Starting Material i Animal Cells 3 x 10 cells Animal Tissues 25 mg for most tissues Blood 100 uL Plasma Serum 200 uL Bacteria 1 x 10 cells Yeast 1 x 10 cells Fungi 50 mg Plant Tissues 50 mg Time to Complete 10 Purifications 22 minutes Average Yields HeLa Cells 1 x 10 cells 15 ug E coli 1 x 10 cells 50 ug 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 Advantages e Fast and easy processing using rapid spin column format Isolate total
4. 2b and 2c as necessary 12 3 Column Wash a Apply 400 uL of 96 100 ethanol provided by the user to the column and centrifuge for 1 minute Note Ensure the entire ethanol 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 spin column with its collection tube c Repeat steps 3a and 3b to wash column a second time d Wash column a third time by adding another 400 uL of 96 100 ethanol provided by the user and centrifuging for 1 minute e Discard the flowthrough and reassemble the spin column with its collection tube f Spin the column for 2 minutes in order to thoroughly dry the resin Discard the collection tube 4 RNA Elution a Place the column into a fresh 1 7 mL Elution tube provided with the kit b Add 50 uL of Elution Solution A to the column c Centrifuge for 2 minutes at 200 x g 2 000 RPM followed by 1 minute at 14 000 x g 14 000 RPM Note the volume eluted from the column If the entire 50 uL has not been eluted spin the column at 14 000 x g 14 000 RPM for 1 additional minute Note For maximum RNA recovery it is recommended that a second elution be performed into a separate microcentrifuge tube Repeat Steps 4b and 4c 5 Storage of RNA The purified RNA sample may be stored at 20 C for a few days It is recommended that samples be pl
5. RNA recovery Ethanol was not Ensure that the appropriate amount of ethanol is added to the added to the lysate lysate before binding to the column Poor RNA Recovery Se sae Ensure that 90 mL of 96 100 ethanol is added to the supplied Wash Solution A prior to use Solution A Different tissues and cells have different RNA contents and thus Low RNA content in the expected yield of RNA will vary greatly from these different cells or tissues used sources Please check literature to determine the expected RNA content of your starting material GellCuliine Cell Ensure that the cell monolayer is washed with the appropriate monolayer was not A k amount of PBS in order to remove residual media from cells washed with PBS Man A Ensure that the appropriate amount of lyticase is added when making the Resuspension Buffer Resuspension Buffer Bacteria and Yeast Ensure that all media is removed prior to the addition of the All traces ofmedia Buffer RL through aspiration not removed 9 p i Insufficient Ensure that the appropriate amount of Buffer RL was used for solubilization of cells the amount of cells or tissue or tissues Clogged Maximum number of ra te cells or amount of Refer to specifications to determine if amount of starting material either tissue exceeds kit falls within kit specifications Genomic DNA specifications Removal Column or High amounts of The lysate may be passed through a 25 gauge needle attached RNA genomic DNA present
6. are changed frequently to avoid contamination e There should be designated solutions tips tubes lab coats pipettes etc for RNA only e All RNA solutions should be prepared using at least 0 05 DEPC treated autoclaved water or molecular biology grade nuclease free water e Clean all surfaces with commercially available RNase decontamination solutions e When working with purified RNA samples ensure that they remain on ice during downstream applications 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 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 Flowchart Procedure for Purifying Total RNA using Norgen s Total RNA Purification Plus Kit A Genomic DNA Removal Lyse cells or tissue using Buffer RL Eliminate DNA using the Genomic DNA W Removal Column SPIN 3 B Purification of RNA A Add ethanol s Bind to RNA Purification i Column Flowthrough with RNA SPIN 3 Wash three times W with Wash Solution A SPIN 4 Elute RNA with W
7. mg Heart 5 mg Kidney 20 mg Liver 20 mg Lung 20 mg Spleen 20 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 RL 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 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 Proceed to Step 2 page 11 1C Lysate Preparation from Blood Notes Prior to Use This procedure is for the isolation of RNA from whole blood For the isolation of RNA from plasma or serum samples please see Appendix A Blood of all human and animal subjects is considered potentially infectious All necessary precautions recommended by the appropriate authorities
8. or tissues Nonetheless these isolated RNA could still be used effectively in different downstream applications such as RT qPCR or microarrays 1 Cell Lysate Preparation from Plasma Serum a Transfer up to 200 uL of plasma or serum to an RNase free microcentrifuge tube not provided b Add 300 uL of Buffer RL to every 100 uL of plasma or serum Mix by vortexing for 10 seconds c Optional Add 0 7 uL of 0 8 ug l MS2 RNA per sample Note The use of MS2 RNA could increase the consistency of downstream applications such as RT PCR However the use of MS2 RNA is not recommended for applications involving global gene expression analysis such as microarrays or sequencing d Add 400 uL of 96 100 ethanol provided by the user to every 400 uL of the lysate equivalent to every 100 uL plasma or serum used Mix by vortexing for 10 seconds Proceed to Step 2 below 2 Binding RNA to Column a Assemble an RNA Purification Column with one of the provided collection tubes b Apply up to 600 uL of the lysate with the ethanol from Step 1 onto the column and centrifuge for 1 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 c Discard the flowthrough Reassemble the spin column with its collection tube d Depending on your lysate volume repeat Step
9. to a syringe 5 10 times in order to shear the genomic DNA prior Purification in sample to loading onto the column Column Centrifuge temperature too low Ensure that the centrifuge remains at room temperature throughout the procedure Temperatures below 15 C may cause precipitates to form that can cause the columns to clog 14 Problem Possible Cause Solution and Explanation RNA is Degraded RNase contamination RNases may be introduced during the use of the kit Ensure proper procedures are followed when working with RNA Please refer to Working with RNA at the beginning of this user guide Procedure not performed quickly enough In order to maintain the integrity of the RNA it is important that the procedure be performed quickly This is especially important for the Cell Lysate Preparation Step in the Animal Tissue protocol since the RNA in animal tissues is not protected after harvesting until it is disrupted and homogenized Improper storage of the purified RNA For short term storage RNA samples may be stored at 20 C for a few days It is recommended that samples be stored at 70 C for longer term storage 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 Starting material may have a h
10. 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 Proceed to Step 2 page 11 1H 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 It is important to work quickly during this procedure 1H 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 b Allow the liquid nitrogen to evaporate without allowing the tissue to thaw c Add 600 uL of Buffer RL to the tissue sample and continue to grind until the sample has been homogenized d Using a pipette transfer the lysate into an RNase free microcentrifuge tube not provided d Spin the lysate for 2 minutes to pellet an
11. Elution Solution A SPIN 4 Purified Total RNA Section 1 Preparation of Lysate From Various Cell Types Notes Prior to Use 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 with the exception of the protocol for plasma serum A separate protocol for the isolation of total RNA from plasma serum samples is located in Appendix B Please ensure that the correct procedure for preparing the lysate from your starting material is followed as indicated in the table below Sample Type Lysate Preparation Page Cultured Cells 5 Animal Tissue 6 Blood 7 Plasma Serum 13 Nasal Throat Swabs 8 Bacteria 8 Yeast 9 Fungi 9 Plant 10 Viruses 10 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 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 Ensure that all solutions are at room temperature prior to use Prepare a working concentration of the Wash Solution A by adding 90 mL of 96 100 ethanol provided by the user to each supplied bottle containing the concentrated Wash Solution A This will give a final volume of 128 m
12. L The label on the bottle has a box that may be checked to indicate that the ethanol has been added 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 and nasal and throat swabs 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 Buffer RL required B mercaptoethanol is toxic and should be dispensed in a fume hood Alternatively the Buffer RL can be used as provided It is important to work quickly during this procedure 1A Lysate Preparation from Cultured Animal Cells Notes Prior to Use The maximum recommended input of cells is 3 x 10 A hemocytometer 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 Buffer RL directly to the frozen cell pellet Step 1A ii c 1A i Cell Lysate Preparation from Cells Growing in a Monolayer a
13. Plus Kit is designed for isolating RNA from small amount of tissue sample up to 20 mg in most cases If a larger amount of starting material is desired Norgen s Animal Tissue RNA Purification Kit Cat 25700 should be used 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 grinding with the mortar and pestle Tissues stored in RNA stabilization reagents such as RNA ater are compatible with this isolation procedure Prior to isolation carefully remove the tissue from the storage reagent using forceps and dry excessive liquid The maximum recommended 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
14. RNA from large rRNA down to microRNA miRNA No phenol or chloroform extractions Isolate high quality total RNA from a variety of sources RNA can be isolated and detected from as little as a single animal cell Rapidly remove contaminating genomic DNA without the use of enzymes Kit Components Component a eee Buffer RL 40 mL 2x40 mL Wash Solution A 38 mL 2x 38 mL Elution Solution A 6 mL 2x6mL RNA Purification Columns 50 100 sort DNA Removal 50 100 Collection Tubes 100 200 Elution tubes 1 7 mL 50 100 Product Insert 1 1 Precautions and Disclaimers This kit is designed for research purposes only Itis 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 The Buffer RL contains guanidinium salts and should be handled with care Guanidinium salts form highly reactive compounds when combined with bleach thus care must be taken to properly dispose of any of these solutions 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 Customer Supplied Reagents and Equipment You must have
15. aced at 70 C for long term storage Related Products Product RNase Free DNase Kit 25710 Animal Tissue RNA Purification Kit 25700 Plant Fungi Total RNA Purification Kit 25800 RNA Protein Purification Kit 24100 RNA DNA Protein Purification Kit 24000 Cytoplasmic amp Nuclear RNA Purification Kit 21000 Leukocyte RNA Purification Kit 21200 microRNA Purification Kit 21300 100b RNA Ladder 15002 1kb RNA Ladder 15003 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 13 Troubleshooting Guide Problem Possible Cause Solution and Explanation Incomplete lysis of Ensure that the appropriate amount of Buffer RL was used for cells or tissue the amount of cells or tissue Either the Genomic DNA Removal Column or the RNA Purification Column may become clogged if the recommended Column has become amount of starting materials is exceeded The amount of starting clogged material may need to be decreased if the column shows clogging below the recommended levels See also Clogged Column below An alternative elution It is recommended that the Elution Solution A supplied with this solution was used kit be used for maximum
16. ex gently and incubate for 5 minutes at room temperature Using a pipette transfer the lysate into another RNase free microcentrifuge tube not provided Note the volume of the lysate Proceed to Step 2 page 11 1E Lysate Preparation from Bacteria Notes Prior to Use Prepare the appropriate lysozyme containing TE Buffer as indicated in Table 2 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 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 1Ec 1E 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
17. for long term storage 11 Appendix A Protocol for Total RNA Purification from Plasma or Serum Notes Prior to Use e Plasma or Serum 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 plasma or serum e We recommend the use of this kit to isolate RNA from plasma or serum prepared by standard protocol from non coagulating fresh blood using EDTA or sodium citrate as the anti coagulant e Plasma prepared from fresh blood using heparin as an anti coagulant is not suitable for use with this protocol For heparin prepared samples follow the protocol in section 1C Lysate Preparation from Blood e Due to the relatively low DNA content in plasma the Genomic DNA Removal column is not necessary for this procedure e It is recommended that no more than 200 uL of plasma or serum be used in order to prevent clogging of the column e Avoid multiple freeze thaw cycle of the plasma or serum sample Aliquot to the appropriate volume for usage prior to freezing e It is important to work quickly during this procedure e The yield of RNA from plasma and serum is highly variable In general the expected yield could vary from 1 to 100 ng per 100 uL plasma or serum used In addition the expected A260 A280 ratio as well as the A260 A230 ratio will be lower lt 1 80 than the normal acceptable range from other cells
18. g DR NORGEN BIOTEK wie CORPORATION 3430 Schmon Parkway Thorold ON Canada L2V 4Y6 Phone 866 667 4362 e 905 227 8848 Fax 905 227 1061 Email techsupport norgenbiotek com Total RNA Purification Plus Kit Product 48300 48400 Product Insert Norgen s Total RNA Purification Plus Kit provides a rapid method for the isolation and purification of total RNA from cultured animal cells tissue samples blood plasma serum bacteria yeast fungi plants and viruses The kit purifies all sizes of RNA from large mRNA and ribosomal RNA down to microRNA miRNA and small interfering RNA siRNA Genomic DNA is removed from the sample using a Genomic DNA Removal column and the RNA is preferentially purified from other cellular components such as proteins without the use of phenol or chloroform 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 Norgen s Purification Technology Purification is based on spin column chromatography The RNA is preferentially purified from other cellular components such as proteins without the use of phenol or chloroform The process involves first lysing the cells or tissue of interest with the provided Buffer RL please see the flow chart on page 4 The DNA is then captured on a Genomic DNA Removal Column Ethanol is
19. igh RNase content For starting materials with high RNAase content it is recommended that B mercaptoethanol be added to the Buffer RL 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 RNA was not washed 3 times with the Traces of salt from the binding step may remain in the sample if the column is not washed 3 times with Wash Solution A Salt RNA does not provided Wash may interfere with downstream applications and thus must be perform well Solution A washed from the column in downstream Ensure that the dry spin under the Column Wash procedure is applications performed in order to remove traces of ethanol prior to elution Ethanol carryover Ethanol is known to interfere with many downstream applications Amount of genomic Genomic DNA DNA in sample Perform additional DNase treatment post isolation Itis contamination exceeds capacity of recommended that Norgen s RNase Free DNase Kit Product Genomic DNA 25710 be used for this step Removal Column 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 P148300 4 M14 15
20. 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 It is important to work quickly during this procedure 1C Cell Lysate Preparation from Blood a b Cc Transfer up to 100 uL of non coagulating blood to an RNase free microcentrifuge tube not provided Add 350 uL of Buffer RL to the blood Lyse cells by vortexing for 15 seconds Ensure that mixture becomes transparent before proceeding to the next step Proceed to Step 2 page 11 1D Lysate Preparation from Nasal or Throat Swabs Notes Prior to Use Body fluid 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 these samples It is important to work quickly during this procedure 1D Cell Lysate Preparation from Nasal or Throat Swabs a b C d e Add 600 uL of Buffer RL to an RNase free microcentrifuge tube not provided Gently brush a sterile single use cotton swab inside the nose or mouth of the subject Using sterile techniques cut the cotton tip where the nasal or throat cells were collected and place into the microcentrifuge tube containing the Buffer RL Close the tube Vort
21. mn has predominately white contents o RNA Purification Columns column has predominately black contents 2 Genomic DNA Removal a Assemble a gDNA Removal Column with one of the provided collection tubes b Apply up to 600 uL of the lysate prepared from Section 1 onto the column and centrifuge at 14 000 x g 14 000 RPM for 1 minute 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 c Retain the flowthrough for RNA Purification Section 3 The flowthough contains the RNA and should be stored on ice or at 20 C until the RNA Purification protocol is carried out d Dispose of the gDNA Removal Column with the bound gDNA 10 Section 3 Total RNA Purification from All Types of Lysate 3 Binding RNA to Column a To every 100 uL of flowthrough from Step 2c add 60 uL of 96 100 Ethanol Mix by vortexing Note For example for 300 uL of flowthrough add 180 uL of 96 100 Ethanol b Assemble an RNA Purification Column with one of the provided collection tubes c Apply up to 600 uL of the lysate with the ethanol onto the column and centrifuge for 1 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
22. of Buffer RL and vortex vigorously for at least 10 seconds Proceed to Step 2 page 11 Table 2 Incubation Time for Different Bacterial Strains 3 Lysozyme Concentration Incubation Time Ee EPRI oe in TE Bufffer Gram negative 1 mg mL 5 min Gram positive 3 mg mL 10 min 1F Lysate Preparation from Yeast Notes Prior to Use Prepare the appropriate amount of Lyticase containing Resuspension Buffer considering that 100 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 uwL 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 1Fc 1F Cell Lysate Preparation a b C d e 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 100 uL of Ly
23. the following in order to use the Total RNA Purification Plus Kit For All Protocols e Benchtop microcentrifuge e 96 100 ethanol e mercaptoethanol optional For Animal Cell Protocol e PBS RNase free For Animal Tissue Protocol e Liquid nitrogen e Mortar and pestle e 70 ethanol For Nasal or Throat Swabs e Sterile single use cotton swabs For Bacterial Protocol e 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 e Resuspension Buffer with Lyticase o 50mM Tris pH 7 5 o 10 mM EDTA o 1 M Sorbital o 1 unit uL Lyticase For Fungi Protocol e Liquid nitrogen e Mortar and pestle e 70 ethanol For Plant Protocol e Liquid nitrogen e Mortar and pestle e 70 ethanol For Plasma Serum Protocol e MS2 RNA 0 8 ug ul Roche Cat No 10165948001 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 e The RNA area should be located away from microbiological work stations e Clean disposable gloves should be worn at all times when handling reagents samples pipettes disposable tubes etc It is recommended that gloves
24. ticase containing Resuspension Buffer by vortexing Incubate at 37 C for 10 minutes Add 300 uL of Buffer RL and vortex vigorously for at least 10 seconds Proceed to Step 2 page 11 1G Lysate Preparation from Fungi Notes Prior to Use Fresh or frozen fungi may be used for this procedure Fungal tissues 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 1G Cell Lysate Preparation from Fungi a b g 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 RL 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
25. y cell debris Transfer the supernatant to another RNase free microcentrifuge tube Note the volume of the supernatant lysate e Proceed to Step 2 page 11 1l Lysate Preparation from Viruses Notes Prior to Use e For the isolation of integrated viral RNA follow Section 1A if the starting material is cell culture follow Section 1B if the starting material is tissue follow Section 1C if the starting material is blood or follow Section 1H if the starting material is a nasal or throat swab e For the isolation of RNA from free viral particles follow the procedure below e It is recommended that no more than 100 uL of viral suspension be used in order to prevent clogging of the column e It is important to work quickly during this procedure 1l Cell Lysate Preparation from Viral Suspension a Transfer up to 100 uL of viral suspension to an RNase free microcentrifuge tube not provided b Add 350 uL of Buffer RL Lyse viral cells by vortexing for 15 seconds Ensure that mixture becomes transparent before proceeding to the next step c Proceed to Step 2 page 11 Section 2 Genomic DNA Removal from All Types of Lysate Notes e The following steps of the procedure for the removal of genomic DNA are the same for all of the different types of lysate e This kit is provided with 2 separate columns When columns are removed from the labelled bags they are supplied in they can easily be identified as follows o gDNA Removal Columns colu
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