E.Z.N.A.® HP Total RNA Kit - Omega Bio-Tek
Contents
1. Digestion Protocol Centrifuge at maximum speed for 2 minutes to completely dry the HiBind RNA Mini Column matrix Note It is important to dry the HiBind RNA Mini Column matrix before elution Residual ethanol may interfere with downstream applications Place the column in a clean 1 5 mL microcentrifuge tube not supplied Add 40 70 uL DEPC Water Note Make sure to add water directly onto the HiBind RNA Mini Column matrix Centrifuge at maximum speed for 2 minutes and store eluted RNA at 70 C Note Any combination of the following steps can be used to help increase RNA yield Heat the DEPC Water to 70 C before adding to the column Increase the incubation time to 5 minutes Increase the elution volume Repeat the elution step with fresh DEPC Water this may increase the yield but decrease the concentration e Repeat the elution step using the eluate from the first elution this may increase yield while maintaining elution volume 25 Troubleshooting Guide Please use this guide to troubleshoot any problems that may arise For further assistance please contact the technical support staff toll free at 1 800 832 8896 i RNA remains on the Repeat the elution ste Little or no RNA colum p p eluted Column is overloaded Reduce the amount of starting material Completely homogenize the sample Incomplete SE Clogged column BE Increase the centrifugation time homogenization Reduce the amount of sta2. Flask Quantification of RNA Quantification and Storage of RNA To determine the concentration and purity of RNA measure absorbance at 260 nm and 280 nm with a spectrophotometer One OD unit measured at 260 nm corresponds to 40 ug mL RNA DEPC Water is slightly acidic and can dramatically lower absorbance values We suggest that you dilute the sample in a buffered solution TE for spectrophotometric analysis The A A ratio of pure nucleic acids is 2 0 while an A A ratio of 0 6 denotes pure protein A ratio of 1 8 2 0 corresponds to 90 100 pure nucleic acid Phenol has a maximum absorbance at 270 nm and can interfere with spectrophotometric analysis of DNA or RNA Store RNA samples at 70 C in water Under these conditions RNA is stable for more than a year Integrity of RNA It is highly recommended that RNA quality be determined prior to beginning all downstream applications The quality of RNA can be best assessed by denaturing agarose gel electrophoresis with ethidium bromide staining The ribosomal RNA bands should appear as sharp clear bands on the gel The 28S band should appear to be double that of the 18S RNA band 235 and 16S if using bacteria If the ribosomal RNA bands in any given lane are not sharp and appear to be smeared towards the smaller sized RNA it is very likely that the RNA undergone degradation during the isolation handling or storage procedure Although RNA molecules less than 200 bases in length do
3. Vacman are all trademarks of their respective companies PCR is a patented process of Hoffman La Roche Use of the PCR process requires a license 27 Notes 28
4. not efficiently bind to the HiBind matrix a third RNA band the tRNA band may be visible when a large number of cells are used Expected Yields For animal cell yields see Page 10 For animal tissue yields see Page 16 Preparing Reagents 1 Dilute RNA Wash Buffer II with 100 ethanol as follows and store at room temperature R6812 02 200 mL 2 Add 20 uL 2 mercaptoethanol B mercaptoethanol per 1 mL GTC Lysis Buffer This mixture can be stored for 4 weeks at room temperature Disruption Techniques for Tissue Samples Efficient sample disruption and homogenization is essential for successful total RNA isolation Cell wall and plasma membrane disruption is necessary for the release of RNA from the sample and homogenization is necessary to reduce the viscosity of the lysates Homogenization shears genomic DNA and other high molecular weight cell components creating a homogeneous lysate Incomplete homogenization can cause the HiBind RNA Mini Column to clog resulting in low or no yield Rotor Stator Homogenizer Using a rotor stator homogenizer for sample disruption can simultaneously disrupt and homogenize most samples The process usually takes less than a minute depending on sample type Many rotor stator homogenizers operate with differently sized probes or generators that allow sample processing in 50 mL tubes Bead Milling By using bead milling cells and tissue can be disrupted by rapid agitation in the presence o
5. the RNA Homogenizer Mini Column 12 10 11 12 13 E Z N A HP Total RNA Kit Animal Cell Protocol Centrifuge at 13 000 x g for 1 minute Save the filtrate and discard the RNA Homogenizer Mini Column Add 1 volume 70 ethanol Vortex to mix thoroughly Do not centrifuge Note A precipitate may form at this point This will not interfere with the RNA purification If any sample has lost its volume during homogenization adjust the volume of ethanol accordingly Insert a HiBind RNA Mini Column into a 2 mL Collection Tube Transfer 700 uL sample including any precipitate that may have formed to the HiBind RNA Mini Column Centrifuge at 10 000 x g for 1 minute Discard the filtrate and reuse the Collection Tube Repeat Steps 10 12 until all of the sample has been transferred to the column Optional This the starting point of the optional on membrane DNase Digestion Protocol Since the HiBind matrix of the RNA Mini Column eliminates most DNA DNase digestion is not necessary for most downstream applications However certain sensitive RNA applications may require further DNA removal If an additional RNA removal step is required please continue to the DNase Digestion Protocol found on Page 25 See DNase Digestion Set E1091 for more information If DNase digestion is not required proceed to Step 14 14 15 16 Add 500 uL RNA Wash Buffer to the HiBind RNA Mini Column Centrifu
6. 1 22 23 18 E Z N A HP Total RNA Kit Animal Tissue Protocol Add 500 uL RNA Wash Buffer to the HiBind RNA Mini Column Centrifuge at 10 000 x g for 30 seconds Discard the filtrate and reuse the Collection Tube Add 500 uL RNA Wash Buffer II to the HiBind RNA Mini Column Note RNA Wash Buffer II must be diluted with ethanol before use Please see Page 8 for instructions Centrifuge at 10 000 x g for 1 minute Discard the filtrate and reuse the Collection Tube Repeat Steps 18 20 for a second RNA Wash Buffer II wash step Centrifuge at maximum speed for 2 minutes to completely dry the HiBind RNA Mini Column Note It is important to dry the HiBind RNA Mini Column matrix before elution Residual ethanol may interfere with downstream applications Transfer the HiBind RNA Mini Column to a clean 1 5 mL microcentrifuge tube not provided 24 25 E Z N A HP Total RNA Kit Animal Tissue Protocol Add 40 70 uL DEPC Water Note Make sure to add water directly onto the HiBind RNA Mini Column matrix Centrifuge at maximum speed for 2 minutes and store eluted RNA at 70 C Note Any combination of the following steps can be used to help increase RNA yield Heat the DEPC Water to 70 C before adding to the column Increase the incubation time to 5 minutes Increase the elution volume Repeat the elution step with fresh DEPC Water this may increase the yield but decrease the concentr
7. 5 16 E Z N A HP Total RNA Kit Animal Tissue Protocol Average Yield of Total Cellular RNA from Mouse Tissue Amount of Tissue mg RNA Yield ug Disrupt the tissue according to one of the following methods described below Amount of GTC Lysis Buffer per Tissue Sample Amount of Tissue Amount of GTC Lysis Buffer uL 20 30 mg 700 uL Note For samples stored in RNALater use 700 uL GTC Lysis Buffer A Rotor Stator Homogenizer Disrupt tissue with a rotor stator homogenizer or until the sample is uniform See Page 9 for details B By using bead milling cells and tissue can be disrupted by rapid agitation in the presence of glass beads and a lysis buffer The optimal size of glass beads to use for RNA isolation are 0 5 mm for yeast unicellular cells and 3 6 mm for animal tissue samples Disrupt according to manufacturers recommended protocol See Page 9 for details Centrifuge at maximum speed for 5 minutes Note In some preparations a fatty upper layer will form after centrifugation Transfer of any of the fatty upper layer may reduce RNA yield or clog the column Insert a RNA Homogenizer Mini Column into a 2 mL Collection Tube Transfer the lysate to the RNA Homogenizer Mini Column 10 11 12 13 14 E Z N A HP Total RNA Kit Animal Tissue Protocol Centrifuge at 13 000 x g for 1 minute Note Make sure that all of the liquid has passed through the RNA Homogenizer Mini Column after
8. A HP Total RNA Kit Animal Cell Protocol 4 Incubate for 3 5 minutes to allow cells to detach Check cells for detachment before proceeding to the next step 5 Add an equal volume of cell culture medium containing serum to inactivate the trypsin 6 Transfer cells to an RNase free glass or polypropylene centrifuge tube not supplied 7 Centrifuge at 500 x g for 5 minutes 8 Aspirate the supernatant 9 Proceed to Step 3 below Note Incomplete removal of the cell culture medium will inhibit lysis and dilute the lysate This will affect the conditions for binding of RNA to the HiBind RNA Mini Column and may reduce RNA yield 3 Disrupt cells do not use more than 1 x 10 cells with GTC Lysis Buffer Vortex or pipet up and down to mix thoroughly Note Add 20 uL 2 mercaptoethanol per 1 mL GTC Lysis Buffer before use Note For pelleted cells loosen the cell pellet thoroughly by flicking the tube before adding the appropriate amount of GTC Lysis Buffer based on the table below To directly lyse the cells in the culture dish add the appropriate amount of GTC Lysis Buffer directly to the dish Collect the cell lysate with a rubber policemen and transfer the cell lysate into a 1 5 mL microcentrifuge tube Number of Cells Amount of GTC Lysis Buffer uL 5x 10 1x 107 700 uL Dish Diameter cm Amount of GTC Lysis Buffer uL 4 Inserta RNA Homogenizer Mini Column into a 2 mL Collection Tube 5 Transfer the lysate to
9. E Z N A HP Total RNA Kit R681 2 00 5 preps R6812 01 50 preps R681 2 02 200 preps March 2015 E Z N A HP Total RNA Kit Table of Contents INrOdUC einen 2 Illustrated Protonen 3 Kit Contents Storage and Stability 4 Important Notes 5 Guidelines for Vacuum Manifold sesssssssssssseeessssssssssseeesees 6 Quantification of RN As 7 Preparing Rega ninas 8 Disruption Techniques for Tissue Samples 9 Animal Cell Protocol ccsesssssssssscssssssssessncsssssscsessnscssssnseacenseees 10 Animal Tissue Protocel eneeeesee en 15 Vacuna Protocol sn 20 DNase Digestion Proton 23 Troubleshooting Guide ae 26 Drei ans 27 Manual Revision March 2015 Fi OMEGA bio tek Innovations in nucleic acid isolation Introduction E Z N A HP Total RNA Kit provides a rapid and easy method for RNA isolation from a small amount of cultured eukaryotic cells or tissues This kit allows single or simultaneous processing of multiple samples in less than 40 minutes Normally 1 x 107 eukaryotic cells or 25 30 mg tissue can be used in a single experiment There is no need for phenol chloroform extractions and time consuming steps such as CsCl gradient ultracentrifugation and precipitation with isopropanol or LiCl are eliminated RNA purified using the E Z N A HP Total RNA method is ready for applications such as RT PCR RT qPCR Northern blotting poly A RNA mRNA purification nuclease protection and in vitro translation Th
10. ase buffers are not compatible with on membrane DNase digestion The use of other buffers may affect the binding of RNA to the HiBind matrix and may reduce RNA yields and purity All steps must be carried out at room temperature Work quickly but carefully 2 Insert the HiBind RNA Mini Column containing the sample into a 2 mL Collection Tube 23 10 11 12 13 14 15 24 Optional DNase Digestion Protocol Add 250 uL RNA Wash Buffer to the HiBind RNA Mini Column Centrifuge at 10 000 x g for 1 minute Discard the filtrate and reuse the Collection Tube Add 75 uL DNase digestion mixture directly onto the surface of the membrane of the HiBind RNA Mini Column Note Pipet the DNase directly onto the membrane DNA digestion will not be complete if some of the mixture is retained on the wall of the HiBind RNA Mini Column Let sit at room temperature for 15 minutes Add 250 uL RNA Wash Buffer to the HiBind RNA Mini Column Let sit at room temperature for 2 minutes Centrifuge at 10 000 x g for 1 minute Discard the filtrate and reuse the Collection Tube Add 500 uL RNA Wash Buffer II Note RNA Wash Buffer II must be diluted with ethanol before use Please see Page 8 for instructions Centrifuge at 10 000 x g for 1 minute Discard the filtrate and reuse the Collection Tube Repeat Steps 12 14 for a second RNA Wash Buffer Il wash step 16 17 18 19 Optional DNase
11. ased on RNA yield and quality obtained from 1 x 10 cells the starting amount can be adjusted for the next purification Source Number of Cells RNA Yield ug E Z N A HP Total RNA Kit Animal Cell Protocol 2 Harvest cells using one of the following methods Do not use more than 1 x 10 cells For cells grown in suspension 1 Determine the number of cells N Centrifuge at 500 x g for 5 minutes 3 Aspirate and discard the supernatant 4 Proceed to Step 3 on Page 12 e For cells grown in a monolayer Note These cells can either be lysed directly in the cell culture dish or trypsinized and collected as a cell pellet prior to lysis Cells grown in cell culture flasks should always be trypsinized For direct cell lysis 1 Determine the number of cells Aspirate and discard the cell culture medium Immediately proceed to Step 3 on Page 12 Note Incomplete removal of the cell culture medium will inhibit lysis and dilute the lysate This will affect the RNA binding conditions of the HiBind RNA Mini Column and may reduce RNA yield To trypsinize and collect cells 1 Determine the number of cells Aspirate and discard the cell culture medium and wash the cells with PBS Note Incomplete removal of the cell culture medium will inhibit trypsin Multiple washes may be necessary for cells that are difficult to detach Add 0 1 0 25 Trypsin in a balanced salt solution 11 E Z N
12. ation e Repeat the elution step using the eluate from the first elution this may increase yield while maintaining elution volume 19 E Z N A HP Total RNA Kit Vacuum Protocol E Z N A Total RNA Kit Protocol Vacuum Protocol All centrifugation steps used are performed at room temperature Materials and Equipment to be Supplied by User Microcentrifuge capable of at least 14 000 x g Vacuum Manifold Vacuum Source RNase free pipet tips and 1 5 mL microcentrifuge tubes 100 ethanol 70 ethanol in sterile DEPC treated water 2 mercaptoethanol B mercaptoethanol Homogenization Equipment Homogenizer Mini Columns HCROO3 Glass Beads Rotor stator Homogenizer Before Starting Prepare GTC Lysis Buffer and RNA Wash Buffer Il according to the Preparing Reagents section on Page 8 Assemble vacuum manifold Please see Page 6 for details Note Please read through previous sections of this manual before proceeding with this protocol Steps 1 8 from the HP Total RNA Animal Cell protocol should be completed or Steps 1 9 from the HP Total RNA Animal Tissue Protocol should be completed before loading the sample to the HiBind RNA Mini Column Instead of continuing with centrifugation follow the steps below Do not use more than 1x10 cells or 10 mg tissue for the vacuum protocol 1 Prepare the vacuum manifold according to manufacturer s instructions 2 Connect the HiBind RNA Mini Column to th
13. centrifugation If necessary repeat the centrifugation until all liquid passes through the membrane Save the filtrate and discard the RNA Homogenizer Mini Column Transfer the cleared lysate to a clean 1 5 mL microcentrifuge tube not supplied Add 1 volume 70 ethanol Vortex to mix thoroughly Do not centrifuge Note A precipitate may form at this point This will not interfere with the RNA purification If any sample has lost its volume during homogenization adjust the volume of ethanol accordingly Insert a HiBind RNA Mini Column into a 2 mL Collection Tube Transfer 700 uL sample including any precipitate that may have formed to the HiBind RNA Mini Column Centrifuge at 10 000 x g for 1 minute Discard the filtrate and reuse the Collection Tube Repeat Steps 11 13 until all of the sample has been transferred to the column Optional This the starting point of the optional on membrane DNase Digestion Protocol Since the HiBind matrix of the RNA Mini Column eliminates most DNA DNase digestion is not necessary for most downstream applications However certain sensitive RNA applications may require further DNA removal If an additional RNA removal step is required please continue to the DNase Digestion Protocol found on Page 25 See DNase Digestion Set Cat E1091 for more information If DNase digestion is not required proceed to Step 15 on the next page 17 13 16 17 18 19 20 2
14. d while maintaining elution volume E Z N A HP Total RNA Kit Animal Tissue Protocol E Z N A HP Total RNA Kit Animal Tissue Protocol All centrifugation steps used are performed at room temperature Materials and Equipment to be Supplied by User Microcentrifuge capable of at least 14 000 x g e RNase free pipette tips RNase free 1 5 mL microcentrifuge tubes 100 ethanol 70 ethanol in sterile DEPC treated water 2 mercaptoethanol B mercaptoethanol Disruption equipment Glass beads Rotor stator homogenizer Before Starting Prepare GTC Lysis Buffer and RNA Wash Buffer Il according to the Preparing Reagents section on Page 8 1 Determine the proper amount of starting material Note It is critical to use the correct amount of tissue in order to obtain optimal yield and purity with the HiBind RNA Mini Column The maximum amount of tissue that can be processed with the Total RNA Protocol is dependent on the tissue type and its RNA content The maximum binding capacity of the HiBind RNA Mini Column is 100 ug The maximum amount of tissue that GTC Lysis Buffer can lyse in the this protocol is 30 mg Use the table on the following page as a guideline to select the correct amount of starting material If no information regarding your starting material is available begin with 10 mg Based on RNA yield and quality obtained from 10 mg the starting amount can be adjusted for the next purification 1
15. e E Z N A HP Total RNA Kit uses the reversible binding properties of HiBind matrix a silica based material This is combined with the speed of mini column spin technology A specifically formulated high salt buffer system allows 100 ug RNA fragments greater than 200 bases to bind to the matrix Cells or tissues are lysed under denaturing conditions that inactivates RNase After the homogenization process by either bead milling or rotor stator homogenizer samples are transferred to a HiBind RNA Homogenizer Mini Column to remove genomic DNA and the filtrate is transferred to a HiBind RNA Mini Column After a few quick washing steps in which cellular debris and other contaminants are effectively washed away high quality RNA is eluted in DEPC Water New in this Edition DNA Clearance Columns have been replaced with RNA Homogenizer Mini Columns Illustrated Protocols Centrifugation Protocol Vacuum Protocol Add GTC Lysis Buffer Add GTC Lysis Buffer and Lyse and Lyse Q Column Column Transfer Lysate to Transfer Lysate to 0 RNA Homogenizer Mini i RNA Homogenizer Mini CN RJ Add Ethanol Add Ethanol Apply Sample to Column Apply Sample to Column Wash 3X J z j Wash 3X Dry Dry Elute Elute Kit Contents R6812 00 R6812 01 en e Storage and Stability All E Z N A HP Total RNA Kit components are guaranteed for at least 12 months from the date of purchase when stored at room temperature During sh
16. e vacuum manifold 3 Load the homogenized sample onto the HiBind RNA Mini Column 20 10 11 12 13 14 E Z N A HP Total RNA Kit Vacuum Protocol Switch on the vacuum source to draw the sample through the column Turn off the vacuum Add 500 uL RNA Wash Buffer to the HiBind RNA Mini Column Switch on the vacuum source to draw the RNA Wash Buffer through the column Turn off the vacuum Add 500 uL RNA Wash Buffer II to the HiBind RNA Mini Column Note RNA Wash Buffer Il must be diluted with ethanol before use Please see Page 8 for instructions Switch on the vacuum source to draw the RNA Wash Buffer through the column Turn off the vacuum Repeat Steps 9 11 for a second RNA Wash Buffer II wash step Transfer HiBind RNA Mini Column to a 2 mL Collection Tube provided with this kit Centrifuge at maximum speed for 2 minutes to completely dry the HiBind RNA Mini Column Note It is important to dry the HiBind RNA Mini Column matrix before elution Residual ethanol may interfere with downstream applications 21 13 16 17 22 E Z N A HP Total RNA Kit Vacuum Protocol Transfer the HiBind RNA Mini Column to a clean 1 5 mL microcentrifuge tube not provided Add 40 70 uL DEPC Water Note Make sure to add water directly onto the HiBind RNA Mini Column matrix Centrifuge at maximum speed for 2 minutes and store eluted RNA at 70 C Note Any combination of the follo
17. f glass beads and a lysis buffer The optimal size of glass beads to use for RNA isolation are 0 5 mm for yeast unicellular cells and 3 6 mm for animal tissue samples E Z N A HP Total RNA Kit Animal Cell Protocol E Z N A HP Total RNA Kit Animal Cell Protocol All centrifugation steps used are performed at room temperature Materials and Equipment to be Supplied by User Microcentrifuge capable of at least 14 000 x g RNase free pipette tips RNase free 1 5 mL microcentrifuge tubes 100 ethanol 70 ethanol in sterile DEPC treated water 2 mercaptoethanol B mercaptoethanol Disruption equipment Glass beads Rotor stator homogenizer Before Starting 1 10 Prepare GTC Lysis Buffer and RNA Wash Buffer Il according to the Preparing Reagents section on Page 8 Determine the proper amount of starting material Note It is critical to use the correct amount of cultured cells in order to obtain optimal yield and purity with the HiBind RNA Mini Column The maximum amount of cells that can be processed with the HP Total RNA Protocol is dependent on the cell line and its RNA content The maximum binding capacity of the HiBind RNA Mini Column is 100 ug The maximum number of cells that GTC Lysis Buffer can efficiently lyse is 1 x 107 Use the following table as a guideline to select the correct amount of starting material If no information regarding your starting material is available begin with 1 x 10 cells B
18. ge at 10 000 x g for 30 seconds Discard the filtrate and reuse the Collection Tube 13 17 18 19 20 21 22 23 24 14 E Z N A HP Total RNA Kit Animal Cell Protocol Add 500 uL RNA Wash Buffer II to the HiBind RNA Mini Column Note RNA Wash Buffer II must be diluted with ethanol before use Please see Page 8 for instructions Centrifuge at 10 000 x g for 1 minute Discard the filtrate and reuse the Collection Tube Repeat Steps 17 19 for a second RNA Wash Buffer II wash step Centrifuge at maximum speed for 2 minutes to completely dry the HiBind RNA Mini Column Note It is important to dry the HiBind RNA Mini Column matrix before elution Residual ethanol may interfere with downstream applications Transfer the HiBind RNA Mini Column to a clean 1 5 mL microcentrifuge tube not provided Add 40 70 uL DEPC Water Note Make sure to add water directly onto the HiBind RNA Mini Column matrix Centrifuge at maximum speed for 2 minutes and store eluted RNA at 70 C Note Any combination of the following steps can be used to help increase RNA yield Heat the DEPC Water to 70 C before adding to the column Increase the incubation time to 5 minutes Increase the elution volume e Repeat the elution step with fresh DEPC Water this may increase the yield but decrease the concentration Repeat the elution step using the eluate from the first elution this may increase yiel
19. ipment crystals or precipitation may form in the GTC Lysis Buffer Dissolve by warming buffer to 37 C Important Notes Please take a few minutes to read this booklet in its entirety to become familiar with the procedures Prepare all materials required before starting to minimize RNA degradation Whenever working with RNA always wear gloves to minimize RNase contamination Use only clean RNase free disposable plastic pipette tips when using the supplied reagents Equilibrate samples and reagents to room temperature before beginning this protocol All steps should be carried out at room temperature unless otherwise noted Work quickly but carefully e Prepare all materials required before starting the procedure to minimize RNA degradation Carefully apply the sample or solution to the center of the HiBind RNA Mini Columns Avoid touching the membrane with pipet tips Guidelines for Vacuum Manifold The following is required for use with the Vacuum Spin Protocol A Vacuum Manifold Compatible Vacuum Manifolds Qiagen QlAvac24 Sigma AldrichVM20 Promega Vacman or manifold with standard Luer connector B Vacuum Flask C Vacuum Tubing D Vacuum Source review tables below for pressure settings Torts Tort Atmospheres atmos 0 000987 Pounds per Square Inch psi 0 0145 Vacuum Setup ream pae ce Kae Vacuum Manifold C Vacuum Tubing D Vacuum Source A Vacuum Manifold B Vacuum
20. rting material Freeze starting material quickly in liquid nitrogen Starting culture Do not store tissue culture cells prior to problems extraction unless they are lysed first Degraded RNA R 9 Follow protocol closely and work quickly Ensure not to introduce RNase during the RNase contamination procedure Check buffers for RNase contamination Ensure RNA Wash Buffer II has been diluted Problem in with 100 ethanol as indicated on bottle Salt carry over durin downstream elution y g RNA Wash Buffer II must be stored and used applications at room temperature Repeat wash steps with RNA Wash Buffer Il Digest with RNase free DNase and inactivate DNA contamination DNase by incubation at 65 C for 5 minutes in the presence of EDTA RNA diluted in acidic DEPC Water is acidic and can dramatically Low Abs ratios lower Abs values Use TE Buffer to dilute buffer or water Fi 2 RNA prior to spectrophotometric analysis DNA contamination 26 Ordering Information The following components are available for purchase separately Call Toll Free at 1 800 832 8896 AA NA RNA Wash Buffer 100 mL RNA Wash Buffer Il 25 mL DEPC Water 100 mL 2 mL Collection Tubes 500 pk 50 pk cs RNase free DNase Set 50 preps RNase free DNase Set 200 preps Proteinase K gt 600 mAU mL Solution 2 mL HiBind E Z N A and MicroElute are registered trademarks of Omega Bio tek Inc Qiagen QlAvac and
21. wing steps can be used to help increase RNA yield Heat the DEPC Water to 70 C before adding to the column Increase the incubation time to 5 minutes Increase the elution volume Repeat the elution step with fresh DEPC Water this may increase the yield but decrease the concentration e Repeat the elution step using the eluate from the first elution this may increase yield while maintaining elution volume Optional DNase Digestion Protocol E Z N A HP Total RNA Kit DNase I Digestion Protocol Since the HiBind matrix of the RNA Mini Column eliminates most DNA DNase digestion is not necessary for most downstream applications However certain sensitive RNA applications may require further DNA removal See DNase Digestion Set Cat E1091 for further information After completing Steps 1 143 of the Animal Cell Protocol Pages 10 13 or Steps 1 14 of the Animal Tissue Protocol Pages 15 17 proceed with the following protocol User Supplied Material DNase Digestion Set E1091 1 For each HiBind RNA Mini Column prepare the DNase stock solution as follows E Z N A DNase Digestion Buffer 73 5 uL RNase free DNase 20 Kunitz uL 1 5 uL Total Volume 75 uL Important Notes DNase lis very sensitive and prone to physical denaturing Do not vortex the DNase mixture Mix gently by inverting the tube Freshly prepare DNase stock solution right before RNA isolation Standard DN
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