Manual - Omega Bio-Tek
Contents
1. Freeze starting material in liquid nitrogen or store sample in RNA Solv Reagent Do not store tissue culture cells prior to extraction unless they are lysed first Follow protocol closely and work quickly Ensure not to introduce RNase during the procedure Check buffers for RNase contamination Problem in downstream applications Salt carry over during elution RNA Wash Buffer II must be diluted with 100 ethanol as instructed on Page 9 RNA Wash Buffer II must be stored and used at room temperature Repeat wash with RNA Wash Buffer II contamination Digest with RNase free DNase and inactivate at 75 C for 5 minutes Low Abs ratios 26 RNA diluted in acidic buffer or water DEPC water is acidic and can dramatically lower Abs260 values Use TE buffer to dilute RNA prior to spectrophotometric analysis Ordering Information The following components are available for purchase separately Call Toll Free at 1 800 832 8896 HiBind E Z N A and MicroElute are registered trademarks of Omega Bio tek Inc Qiagen QlAvac and 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 282. To trypsinize and collect cells 1 Determine the number of cells 2 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 3 Add 0 1 0 25 Trypsin in a balanced salt solution 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 Aspirate the supernatant 9 Proceed to Step 3 on Page 12 g 11 10 11 12 13 12 E Z 96 Total RNA Kit Il Cultured Cells Protocol Add 800 uL RNA Solv Reagent Vortex or pipet up and down to mix thoroughly Important RNA Solv Reagent must be mixed with 2 mercaptoethanol before use Please see Page 9 for instructions Note For pelleted cells loosen the cell pellet thoroughly by flicking the tube before adding RNA Solv Reagent To directly lyse the cells in the culture dish add RNA Solv Reagent directly to the dish Proceed to Step 4 below Collect the cell lysates with a rubber policemen and transfer to a 96 well deep well plate not provided Let sit at room temperature for 5 minutes Add 160 uL chloroform to each sam
3. Note Any combination of the following steps can be used to help increase RNA yield Preheat the DEPC Water to 70 C before adding to the column Increase the incubation time to 5 minutes e Increase the elution volume 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 yield while maintaining elution volume Store eluted RNA at 70 C E Z 96 Total RNA Kit Il Animal Tissue Protocol E Z 96 Total RNA Kit II Protocol Animal Tissue Protocol Materials and Equipment to be Supplied by User Centrifuge capable of 4 000 x g with 96 well plate adaptor RNase free pipet tips Multichannel pipette e Disposable reservoirs 96 well Deep well Plates 1 or 2 mL e 14 3M 2 mercaptoethanol B mercaptoethanol e Chloroform 100 ethanol 70 ethanol in sterile DEPC treated water Homogenization Equipment Omega Homogenizer Plates HCR9601 e Needle and syringe Mortar and pestle Glass beads Rotor stator homogenizer Before Starting Prepare RNA Wash Buffer II and RNA Solv Reagent according to the Preparing Reagents section on Page 9 1 Determine the proper amount of starting material Note It is critical to use the correct number of cells to obtain optimal yield and purity with the E Z 96 RNA Plate The maximum amount of tissue that can be processed wi
4. Plate Switch on the vacuum source to draw the RNA Wash Buffer through the column Turn off the vacuum Add 750 uL RNA Wash Buffer II to the E Z 96 RNA Plate Note RNA Wash Buffer II 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 Repeat Steps 8 10 for a second RNA Wash Buffer II wash step Continue to apply the vacuum for 15 minutes after all liquid has passed through the E Z 96 RNA Plate Note It is important to dry the E Z 96 RNA Plate matrix before elution Residual ethanol may interfere with downstream applications Transfer the E Z 96 RNA Plate to a 96 well Microplate provided with this kit Add 40 70 uL DEPC Water to each well of the E Z 96 RNA Plate Note Make sure to add water directly onto the E Z 96 RNA Plate matrix 21 15 16 17 18 22 E Z 96 Total RNA Kit Il Vacuum Protocol Let sit at room temperature for 1 minute Centrifuge at 4 000 x g for 3 minutes Repeat Steps 23 25 for a second elution step Note Any combination of the following steps can be used to help increase RNA yield e Preheat 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 firs
5. E Z 96 Total RNA Kit II Table of Contents Introduction and OVEFVIEW cscsssccssecsscssecseessecsecessecneceneersees 2 Kit Contents Storage and Stability secsecsssecsecseeeneers 3 Important NOES assess sete cetacean eee 4 Tissue Homogenization TECHNIQUES c ssssssseseeseesseseeseeens 5 Quantification of RNA ssssssseessseeeesreeresrseesereeessreeeeereesrreeesrrsees 7 Guideline for Vacuum Manifold ssssssssssseesssrsssssssssseseesssssss 8 Preparing Reagents essssesssserssseerssseresseeessseesssseoossesssssossssessss 9 Cultured Cell ProtOCoOl essssssseeessssssssseseeeseessssssssseeseessnssnsssessee 10 Animal Tissue ProtoCol ssesssssssssssessessssssssssssceessrsssssessreeeress 15 Vacu m PROTO CO esic ccinncsinmaeandanemnnicawannese 20 DNase I Digestion PIOLOCG hivscesctcctesreerereis creme 23 Troubleshooting Guide sssssssssesessssssssssteeseesssssssseeeserssesssss 26 Orderin Ganie a AARE RAEAN GEAR 27 Manual Revision August 2012 02 OMEGA bio tek Innovations in nucleic acid isolation Introduction and Overview E Z 96 Total RNA Kit Il is designed to isolate total cellular RNA from all types of animal and human tissues This kit allows simultaneous purification of 96 samples in less than 60 minutes RNA purified using this kit is ready for applications such as RT PCR Northern blotting poly A RNA mRNA purification nuclease protection and in vitro translation The E Z 96 Tot
6. a homogenizer plate placed on a 96 well deep well plate not provided Centrifuge at maximum speed for 5 minutes Save the filtrate Discard the plate Note Incomplete homogenization of the sample may cause the column to clog resulting in decreased yield It is recommended to homogenize the tissue samples with a rotor stator homogenizer as this method normally produces better yields 3 Let sit at room temperature for 5 minutes 4 Transfer the tissue lysates to a 96 well deep well plate not provided 16 10 11 12 13 14 E Z 96 Total RNA Kit Il Animal Tissue Protocol Add 160 uL chloroform Vortex to mix thoroughly Let sit at room temperature for 2 3 minutes Seal the plate with Sealing Film Centrifuge at gt 4 000 x g for 20 minutes at 4 C to separate the aqueous and organic phase Note The sample should separate into 3 phases an upper colorless aqueous phase which contains RNA a white interphase and a lower blue organic phase Transfer the upper aqueous phase 350 400 uL into a new 96 well deep well plate Add 1 volume 70 ethanol to each well Pipet up and down 20 30 to mix thoroughly A precipitate may form at this point This will not interfere with the RNA purification Place an E Z 96 RNA Plate on top of a 96 well Square well Plate supplied Transfer the samples to the E Z 96 RNA Plate and seal the plate with AeraSeal Film Centrifuge at 4 000 x g for 5 minutes at room temperature Re
7. al RNA Kit II provides a fast and easy method for high throughput RNA isolation This kit integrates efficient lysis of a phenol guanidine buffer and the reversible binding properties of HiBind matrix a silica based material with the speed of the E Z 96 plate format for fast processing of large sample numbers Cells or tissues are homogenized with RNA Solv Reagent that inactivates RNases Chloroform is added to separate the homogenate into aqueous and organic phases via centrifugation The aqueous phase which contains RNA is adjusted with ethanol and then applied to the E Z 96 RNA Plate to bind total RNA while cellular debris and other contaminants are effectively washed away High quality RNA is eluted in DEPC Water New in this Edition This manual has been edited for content and redesigned to enhance user readability Kit Contents Purifications E Z 96 RNA Plate 4 96 well Square well Plate 2 2 mL 1 4 96 well Microplate 500 uL 1 4 1 4 3 1 Sealing Film To Aera Seal Film 2 RNA Solv Reagent 4x25 mL 4x 100 mL RNA Wash Buffer 360 mL RNA Wash Buffer II 140 mL DEPC Water 60 mL User Manual v Warning RNA Solv Reagent contains guanidine thiocyanate and phenol Handle with care Storage and Stability All of the E Z 96 Total RNA Kit II components are guaranteed for at least 12 months from the date of purchase when stored as follows RNA Solv Reagent should be stored at 2 8 C for long term use All othe
8. am applications Transfer the E Z 96 RNA Plate to a 96 well Microplate provided Add 40 70 uL DEPC Water to each well of the E Z 96 RNA Plate Note Make sure to add water directly onto the E Z 96 RNA Plate matrix Let sit at room temperature for 1 minute Centrifuge at 4 000 x g for 3 minutes Repeat Steps 18 20 for a second elution step Note Any combination of the following steps can be used to help increase RNA yield Preheat 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 Store eluted RNA at 70 C 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 Problem Little or no RNA eluted RNA remains on the column Repeat elution Preheat DEPC Water to 70 C prior to elution Incubate column for 10 minutes with water prior to centrifugation Column is overloaded Reduce quantity of starting material Incomplete homogenization Completely homogenize sample Increase centrifugation time Reduce amount of starting material Degraded RNA RNase contamination
9. ars Multiply by Millimeters of Mercury mmHg 0 75 Kilopascals kPa 0 1 Inches of Mercury inchHg 0 0295 Tors Tor Atmospheres atmos 0 000987 Pounds per Square Inch psi 0 0145 Illustrated Vacuum Setup a _S Omega Bio tek s VAC 03 C Vacuum Tubing D Vacuum Source A Vacuum Manifold B Vacuum Flask Preparing Reagents Dilute RNA Wash Buffer II with 100 ethanol as follows and store at room temperature R6935 00 140 mL R6935 01 560 mL R6935 02 640 mL per bottle Add 20 uL 2 mercaptoethanol per 1 mL RNA Solv Reagent E Z 96 Total RNA Kit Il Cultured Cells Protocol E Z 96 Total RNA Kit II Protocol Cultured Cell Protocol Materials and Equipment to be Supplied by User Centrifuge capable of 4 000 x g with 96 well plate adaptor RNase free pipet tips Multichannel pipette Disposable reservoirs 96 well deep well plates 1 or 2 mL 14 3M 2 mercaptoethanol 8 mercaptoethanol Chloroform 100 ethanol 70 ethanol in sterile DEPC treated water Before Starting 10 Prepare RNA Wash Buffer Il and RNA Solv Reagent according to the Preparing Reagents section on Page 9 Determine the proper amount of starting material Note It is critical to use the correct number of cells to obtain optimal yield and purity with the E Z 96 RNA Plate The maximum amount of cells that can be processed with the Total RNA Protocol is dependent on the cell line and its RNA content The
10. ips Multichannel pipette e Disposable reservoirs 96 well Deep well Plates 1 or 2 mL 14 3M 2 mercaptoethanol B mercaptoethanol e Chloroform 100 ethanol e 70 ethanol in sterile DEPC treated water e Homogenization Equipment for Animal Tissue Protocol Omega Homogenizer Plates HCR9601 e Needle and syringe e Mortar and pestle Glass beads Rotor stator homogenizer Before Starting Prepare RNA Wash Buffer II and RNA Solv Reagent according to the Preparing Reagents section on Page 9 Assemble vacuum manifold see Page 8 for details Note Please read through previous sections of this manual before proceeding with this protocol Steps 1 11 from the Cultured Cell Protocol should be completed or Steps 1 10 from the Animal Tissue Protocol should be completed before loading the sample to the E Z 96 RNA Plate 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 E Z 96 RNA Plate to the vacuum manifold 20 10 11 12 13 14 E Z 96 Total RNA Kit Il Vacuum Protocol Transfer the samples from Step 11 Cultured Cell Protocol or Step 10 Animal Tissue Protocol to the E Z 96 RNA Plate Switch on the vacuum source to draw the sample through the column Turn off the vacuum Add 500 uL RNA Wash Buffer to the E Z 96 RNA
11. maximum binding capacity for each well in the E Z 96 RNA Plate is 100 ug The maximum number of the cells that RNA Solv Reagent can efficiently lyse is 1x107 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 1 x 10 cells Based on RNA yield and quality obtained from 1 x 10 cells the starting amount can be adjusted for the next purification Average Yield of Total Cellular RNA Number of Cells RNA Yield ug E Z 96 Total RNA Kit Il Cultured Cells Protocol Harvest Cells 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 E Z 96 RNA Plate and may reduce RNA yield A For cells grown in suspension 1 Count cells 2 Transfer 1 x 10 cells no more than 1 x 10 cells toa 15 mL centrifuge tube 3 Centrifuge at 500 x g for 5 minutes Aspirate and discard the media 5 Continue to Step 3 on Page 12 gt A B For cells grown in a mono layer 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 e For direct cell lysis 1 Determine the number of cells 2 Aspirate and discard the cell culture medium 3 Immediately proceed to Step 3 on Page 12
12. move and discard the AeraSeal Film Discard the filtrate and reuse the 96 well Square well Plate Optional This the starting point of the optional on membrane DNase Digestion Protocol Since the HiBind matrix of the E Z 96 RNA Plate 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 23 See DNase Digestion Set E1091 for more information If DNase digestion is not required proceed to Step 15 17 15 16 17 18 19 20 21 22 23 24 25 18 E Z 96 Total RNA Kit II Animal Tissue Protocol Add 600 uL RNA Wash Buffer to each well of the E Z 96 RNA Plate and seal the plate with AeraSeal Film Centrifuge at 4 000 x g for 5 minutes at room temperature Remove and discard the AeraSeal Film Discard the filtrate and reuse the 96 well Square well Plate Add 750 uL RNA Wash Buffer II to each well of the E Z 96 RNA Plate and seal the plate with AeraSeal Film Note RNA Wash Buffer II must be diluted with ethanol before use Please see Page 9 for instructions Centrifuge at 4 000 x g for 5 minutes at room temperature Remove the AeraSeal Film Discard the filtrate and reuse the 96 well Square well Plate Repeat Steps 18 20 for a second RNA Wash Buffer II wash step Ce
13. nto a 96 well Square well Plate 23 10 11 12 13 14 15 24 E Z 96 Total RNA Kit II DNase I Digestion Protocol Add 300 uL RNA Wash Buffer to each well of the E Z 96 RNA Plate Let sit at room temperature for 2 minutes Centrifuge at 4 000 x g for 3 minutes Add 75 uL DNase digestion mixture directly onto the surface of the membrane of the E Z 96 RNA Plate 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 E Z 96 RNA Plate Let sit at room temperature for 15 minutes Add 300 uL RNA Wash Buffer I to each well of the E Z 96 RNA Plate Let sit at room temperature for 2 minutes Centrifuge at 4 000 x g for 3 minutes Discard the filtrate and reuse the 96 well Square well Plate Add 750 uL RNA Wash Buffer II to each well of the E Z 96 RNA Plate Note RNA Wash Buffer II must be diluted with ethanol before use Please see Page 8 for instructions Centrifuge at 4 000 x g for 3 minutes Discard the filtrate and reuse the 96 well Square well Plate Repeat Steps 12 14 for a second RNA Wash Buffer II wash step 16 17 18 19 20 21 22 E Z 96 Total RNA Kit II DNase I Digestion Protocol Centrifuge at maximum speed for 20 minutes to completely dry the E Z 96 RNA Plate matrix Note It is important to dry the E Z 96 RNA Plate matrix before elution Residual etha nol may interfere with downstre
14. ntrifuge at maximum speed for 20 minutes to completely dry the E Z 96 RNA Plate matrix Note It is important to dry the E Z 96 RNA Plate matrix before elution Residual ethanol may interfere with downstream applications Transfer the E Z 96 RNA Plate to a 96 well Microplate provided Add 40 70 uL DEPC Water to each well of the E Z 96 RNA Plate Note Make sure to add water directly onto the E Z 96 RNA Plate matrix Let sit at room temperature for 1 minute 26 27 28 E Z 96 Total RNA Kit Il Animal Tissue Protocol Centrifuge at 4 000 x g for 3 minutes Repeat Steps 24 26 for a second elution step Note Any combination of the following steps can be used to help increase RNA yield Preheat 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 Store eluted RNA at 70 C 19 E Z 96 Total RNA Kit Il Vacuum Protocol E Z N A Total RNA Kit Protocol Vacuum Method All centrifugation steps used are performed at room temperature Materials and Equipment to be Supplied by User Vacuum Manifold e Vacuum Source e Centrifuge capable of 4 000 x g with 96 well plate adaptor RNase free pipet t
15. on 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 E Z 96 RNA Plate to clog resulting in low or no yield Liquid Nitrogen Method 1 Wear appropriate gloves and take great care when working with liquid nitrogen 2 Excise tissue and promptly freeze in a small volume of liquid nitrogen 3 Grind tissue with a ceramic mortar and pestle under approximately 10 mL liquid nitrogen 4 Pour the suspension into a pre cooled 15 mL polypropylene tube Note Unless the tube is pre cooled in liquid nitrogen the suspension will boil vigorously and may cause loss of tissue 5 Allow the liquid nitrogen to completely evaporate and add RNA Solv Reagent 6 Proceed to one of the homogenization steps below Choose a homogenization step below 1 Omega Homogenizer Plates HCR9601 01 or HCR9601 02 Load the lysate into a homogenizer plate placed on a 96 well deep well plate not provided Centrifuge at maximum speed for 5 minutes Save the filtrate and discard the plate Proceed to Step 1 of the Animal Tissue Protocol on Page 15 2 Syringe and Needle Shear high molecular weight DNA by passing the lysate through a narr
16. ow needle 19 21 gauge 5 10 times Save the lysate Proceed to Step 1 of the Animal Tissue Protocol on Page 15 Tissue Homogenization Techniques Rotor Stator Homogenizer Sample Disruption and Homogenization Using a rotor stator homogenizer for sample disruption and homogenization 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 Sample Disruption and Homogenization By using bead milling cells and tissue can be disrupted and homogenized 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 4 8 mm for animal tissue samples 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 g 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 ha
17. ple Pipet up and down to mix thoroughly Let sit at room temperature for 2 3 minutes Seal the plate with Sealing Film Centrifuge at 4 000 x g for 20 minutes at 4 C to separate the aqueous and organic phase Note The sample should separate into 3 phases an upper colorless aqueous phase which contains RNA a white interphase and a lower blue organic phase Transfer the upper aqueous phase 350 400 uL into a new 96 well deep well plate Add 1 volume 70 ethanol to each well Pipet up and down 20 30 to mix thoroughly A precipitate may form at this point This will not interfere with the RNA purification Place an E Z 96 RNA Plate on top of a 96 well Square well Plate supplied Transfer the samples to the E Z 96 RNA Plate and seal the plate with AeraSeal Film E Z 96 Total RNA Kit Il Cultured Cells Protocol 14 Centrifuge at 4 000 x g for 5 minutes at room temperature Remove and discard the AeraSeal Film 15 Discard the filtrate and reuse the 96 well Square well Plate Optional This the starting point of the optional on membrane DNase Digestion Protocol Since the HiBind matrix of the E Z 96 RNA Plate 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 23 See DNase Digestion Set E1091 for mo
18. r components should be stored at room temperature 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 e 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 Carefully apply the sample or solution to the center of each well of the E Z 96 RNA Plate Avoid touching the membrane with pipet tips For long term storage of whole tissue flash freeze samples in liquid nitrogen and immediately transfer to 70 C Tissue can be stored for up to 6 months at 70 C To process frozen tissue samples do not thaw the sample during weighing or handing prior to the disruption with RNA Solv Reagent Tissue samples homogenized in RNA Solv Reagent can be stored at 70 C for at least 6 months To process frozen tissue homogenates thaw the sample at 37 C until it is completely thawed and all salts in the lysis buffer are dissolved Do not extend the treatment in 37 C because it can cause chemical degradation of RNA Tissue Homogenization Techniques Efficient tissue sample disruption and homogenizati
19. re information If DNase digestion is not required proceed to Step 16 16 Add 600 uL RNA Wash Buffer I to each well of the E Z 96 RNA Plate and seal the plate with AeraSeal Film 17 Centrifuge at 4 000 x g for 5 minutes at room temperature Remove and discard the AeraSeal Film 18 Discard the filtrate and reuse the 96 well Square well Plate 19 Add 750 uL RNA Wash Buffer II to each well of the E Z 96 RNA Plate and seal the plate with AeraSeal Film Note RNA Wash Buffer II must be diluted with ethanol before use Please see Page 9 for instructions 20 Centrifuge at 4 000 x g for 5 minutes at room temperature Remove the AeraSeal Film 21 Discard the filtrate and reuse the 96 well Square well Plate 22 Repeat Steps 19 21 for a second RNA Wash Buffer II wash step 13 23 24 25 26 27 28 29 14 E Z 96 Total RNA Kit Il Cultured Cells Protocol Centrifuge at maximum speed for 20 minutes to completely dry the E Z 96 RNA Plate matrix Note It is important to dry the E Z 96 RNA Plate matrix before elution Residual ethanol may interfere with downstream applications Transfer the E Z 96 RNA Plate to a 96 well Microplate provided Add 40 70 uL DEPC Water to each well of the E Z 96 RNA Plate Note Make sure to add water directly onto the E Z 96 RNA Plate matrix Let sit at room temperature for 1 minute Centrifuge at 4 000 x g for 3 minutes Repeat Steps 25 27 for a second elution step
20. s 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 23S 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 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 Guideline for Vacuum Manifold The following is required for use with the Vacuum Protocol A Vacuum Manifold We recommend Omega Bio tek s VAC 03 Other 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 Recommended Pressure mbar VAC 03 200 to 400 Conversion from millib
21. t elution this may increase yield while maintaining elution volume Store eluted RNA at 70 C E Z 96 Total RNA Kit II DNase I Digestion Protocol E Z N A 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 15 of the Cell Cultured 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 I Digestion Set E1091 1 For each E Z 96 RNA Plate prepare the DNase I 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 DNase 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 Place the E Z 96 RNA Plate containing the sample o
22. th the Total RNA Protocol is dependent on the type of tissue and its RNA content The maximum binding capacity for each well in the E Z 96 RNA Plate is 100 ug The maximum amount of tissue that RNA Solv Reagent can efficiently lyse is 50 mg For liver or spleen only 25 mg tissue should be used 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 tissue Based on RNA yield and quality obtained from 10 mg the starting amount can be adjusted for the next purification 15 E Z 96 Total RNA Kit II Animal Tissue Protocol Average Yield of Total Cellular RNA Mouse Tissue Sample Amount of Tissue RNA Yield ug mg 2 Homogenize and disrupt the tissue in 800 uL RNA Solv Reagent according to one of the following methods described below Do not let the sample to thaw before adding the RNA Solv Reagent Important RNA Solv Reagent must be mixed with 2 mercaptoethanol before use Please see Page 9 for instructions A Rotor Stator Homogenizer Homogenize tissue with a rotor stator homogenizer until the sample is uniformly homogenized See Page 6 for details B Liquid Nitrogen Method See Page 5 for detailed protocol 1 Syringe and Needle Shear high molecular weight DNA by passing the lysate through a narrow needle 19 21 gauge 5 10 times 2 Omega Homogenizer Plate HCR9601 Load the lysate into
Download Pdf Manuals
Related Search