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Manual - Omega Bio-Tek

1. 50 mL Collection Tube 13 18 19 20 21 22 23 24 12 E Z N A Ultra Pure Total RNA Maxi Kit Protocols Centrifuge at 5 000 x g for 5 minutes Discard the filtrate and reuse the collection tube Repeat Steps 17 19 for a second RNA Wash Buffer II wash step Centrifuge the empty column at 5 000 x g for 10 minutes to completely dry the HiBind matrix Note It is important to dry the HiBind RNA Maxi Column matrix before elution Residual ethanol may interfere with downstream applications Transfer the HiBind RNA Maxi Column to a clean 50 mL centrifuge tube not supplied Add 1 3 mL DEPC Water Make sure to add the water directly to the center of the column matrix Centrifuge at 5 000 x g for 5 minutes A second elution may be necessary if the expected yield of RNA gt 2 mg 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 Let sit at room temperature for 5 minutes after adding DEPC Water e Increase the elution volume e 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 E Z N A Ultra Pure Total RNA Maxi Kit Protocols E Z N A Ultra Pure Total RNA Maxi Kit Animal Tissue Protocol All centrifugation
2. RNA Maxi Kit utilizes the reversible binding properties of the HiBind matrix a new silica based material By combining the high lysis efficiency of RNA Solv Reagent with Omega Bio tek s innovative HiBind technology this kit can extract total cellular RNA from all types of animal or human tissues including fatty tissues such as brain and adipose tissue A specifically formulated high salt buffer system allows more than 5 mg RNA molecules greater than 200 bases to bind to the matrix Cells or tissue are homogenized with RNA Solv Reagent that inactivates RNases After the addition of chloroform the homogenate is separated into aqueous and organic phases The aqueous phase which contains the RNA is adjusted with ethanol and applied to the HiBind RNA Maxi Column The HiBind matrix binds total RNA while cellular debris and other contaminants are effectively washed away High quality RNA is eluted in DEPC Water Binding Capacity Each HiBind RNA Maxi Column can bind approximately 5 mg total RNA Using greater than 1 g tissue or 2 g adipose tissue is not recommended New in this Edition This manual has been edited for content and redesigned to enhance user readability 19 Notes 18 Illustrated Protocol Add RNA Solv Reagent Homogenize Tissue Add Chloroform Gn O Transfer Aqueous Phase T fl Add Ethanol Ww j Apply Sample to Column N E d Wash 3x 7 Dry Elute Kit Contents maa
3. 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 Homogenization Choose one method below 1 Homogenizer Spin Columns and 15 mL Collection Tubes Load the lysate into a homogenizer spin column pre inserted into a 15 mL Collection Tube not provided Spin for 5 minutes at maximum speed in a centrifuge in order to collect homogenized lysate Proceed to Step 1 of the Animal Tissue Protocol on Page 9 2 Syringe and Needle Shear high molecular weight DNA by passing the lysate through a narrow needle 19 21 gauge 5 10 times Proceed to Step 1 of the Animal Tissue Protocol on Page 9 Disruption and Homogenization of Samples 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 ly
4. 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 Maxi 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 13 See DNase Digestion Set Cat E1091 for more information If DNase digestion is not required proceed to Step 14 14 Add 16 mL RWF Wash Buffer 15 Centrifuge at 5 000 x g for 5 minutes 16 Discard the filtrate and reuse the collection tube 17 Add 10 mL RNA Wash Buffer Il Note RNA Wash Buffer Il must be diluted with ethanol before use Please see Page 8 for instructions 11
5. Notes 20 E Z N A Ultra Pure Total RNA Maxi Kit Table of Contents Hacicess Ohad co g Meereerarenr Seeenen ice Sete om t tort niey Narre tee tonne 2 Nis Creat Protocolar 3 Kit Contents Storage and Stability essssesssssceseeeeees 4 Preparing REAGEMNS Arcisnsinnevannannsionedmmnetneiasnsiens 3 Quantification of RNA sss sssssssssssssssereesssssssssseceresssssssssserreeess 6 Disruption and Homogenization ssssssssssssseeeesssssssssseee 7 Animal Tissue ProtoCol ssssssssssssesesssssssssssccersssssssssssseeeeeeensssss 9 DNase Digestion Protocoll ssssssssssscsscsecssecssecsserseerseerss 13 Troubleshooting Guide ssesssssssssesssssssssssseeeeessssssssseerresssss 16 ORMEHING e seeressesi es rn EE PEE E EN Ea 17 Manual Revision March 2012 N OMEGA bio tek Innovations in nucleic acid isolation Introduction Notes The E Z N A Ultra Pure Total RNA Maxi Kit is designed to isolate total cellular RNA from tissues rich in triglycerides and fatty acids such as brain and adipose tissues However this kit can also be used for the isolation of total cellular RNA from other type of tissues including cultured eukaryotic cells animal tissues or bacteria RNA purified using the E Z N A Ultra Pure Total RNA Maxi method is ready for applications such as RT PCR Northern blotting poly A RNA mRNA purification nuclease protection assay and in vitro translation The E Z N A Ultra Pure Total
6. ater Under these conditions RNA is stable for more than a year To freeze tissue for long term storage flash freeze tissue in liquid nitrogen and immediately transfer to 70 C Tissue can be store for up to 6 months at 70 C To process the sample do not thaw the sample during weighing or handing prior to the disruption with RNA Solv Reagent Homogenized tissue lysates can be store at 70 C for at least 6 months To proceed with the frozen tissue lysates thaw the sample at 37 C until they are 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 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 us
7. e contamination procedure Check buffers for RNase contamination Dilute RNA Wash Buffer II with 100 ethanol Problem in as instructed on Page 5 downstream ae oa during RNA Wash Buffer II must be stored and used applications at room temperature Repeat wash step with RNA Wash Buffer II DNA ee Follow the DNase Digestion Protocol on eer DNA contamination contamination Page 13 Problem Cause Solution __ 4 DEPC Water is acidic and can dramatically x RNA diluted in acidic f Low Abs ratios lower Abs values Use TE buffer to dilute buffer or water eee RNA prior to spectrophotometric analysis 16 Preparing Reagents Dilute RNA Wash Buffer II with 100 ethanol as follows and store at room temperature Oooo o O ooa 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 w
8. ed 16 17 18 19 E Z N A Ultra Pure Total RNA Maxi Kit Protocols Centrifuge the empty column at 5 000 x g for 10 minutes to completely dry the HiBind matrix Note It is important to dry the HiBind RNA Maxi Column matrix before elution Residual ethanol may interfere with downstream applications Transfer the HiBind RNA Maxi Column to a clean 50 mL centrifuge tube not supplied Add 1 3 mL DEPC Water Note Make sure to add water directly onto the HiBind RNA Maxi Column matrix Centrifuge at 5 000 x g for 5 minutes and store eluted RNA at 70 C 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 Let sit at room temperature for 5 minutes after adding DEPC Water Increase the elution volume e 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 15 10 11 12 13 14 15 14 E Z N A Ultra Pure Total RNA Maxi Kit Protocols Add 8 mL RWF Wash Buffer Centrifuge at 5 000 x g for 10 minutes Discard the filtrate and reuse the collection tube Add 1 5 mL DNase digestion mixture directly onto the surface of the membrane of the HiBind RNA Maxi Column Note Pipet the DNase directly onto the
9. membrane DNA digestion will not be complete if some of the mixture is retained on the wall of the HiBind RNA Maxi Column Let sit at room temperature for 15 minutes Add 8 mL RWF Wash Buffer Let sit at room temperature for 2 minutes Centrifuge at 5 000 x g for 5 minutes Discard the filtrate and reuse the collection tube Add 10 mL RNA Wash Buffer Il Note RNA Wash Buffer II must be diluted with ethanol before use Please see Page 5 for instructions Centrifuge at 5 000 x g for 5 minutes Discard the filtrate and reuse the collection tube Repeat Steps 12 14 for a second RNA Wash Buffer II wash step Disruption and Homogenization of 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 homogenous lysate Incomplete homogenization can cause the HiBind RNA Maxi Column 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
10. oo ra RR Storage and Stability All components in the Ultra Pure Total RNA Maxi Kit except the RNA Solv Reagent should be stored at room temperature RNA Solv Reagent should be store at 2 8 C All Ultra Pure Total RNA Maxi Kit components are guaranteed for at least 12 months from the date of purchase when stored at the indicated temperatures Ordering Information The following components are available for purchase separately Call Toll Free at 1 800 832 8896 C rrr DEPC Water 100 mL PRO32 RNase free DNase Set 1500 units E1091 RNase free DNase Set 6000 units E1091 02 HiBind E Z N A and MicroElute are registered trademarks of Omega Bio tek Inc PCR is a patented process of Hoffman La Roche Use of the PCR process requires a license 17 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 RNA remains on the luti Little or no column Repeat elution step RNA eluted Column is overloaded Reduce quantity of starting material Completely homogenize sample Clogged Incomplete Saas 99 sae Increase centrifugation time column homogenization Reduce amount of starting material i Freeze starting material quickly in liquid Starting material nitrogen problems Follow protocol closely and work quickly Degraded RNA i Ensure not to introduce RNase during the RNas
11. sis 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 E Z N A Ultra Pure Total RNA Maxi Kit Protocols E Z N A Ultra Pure Total RNA Kit DNase I Digestion Protocol Since the HiBind matrix of the RNA Maxi 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 13 of the Animal Tissue Protocol Pages 9 11 proceed with the following protocol User Supplied Material DNase Digestion Set Cat E1091 1 For each HiBind RNA Maxi Column prepare the DNase stock solution as follows E Z N A DNase Digestion Buffer 1 47 mL RNase free DNase 20 Kunitz uL 30 uL Important Notes e 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 Insert the HiBind RNA Maxi Column containing the sample into a
12. ssues source Amount ofrisue mg _RNAVieKI a O wn wo f o O omn wo o e a E SCS Disrupt and homogenize the tissue in 20 mL RNA Solv Reagent according to one of the following methods described on Pages 7 8 Note Incomplete homogenization of the sample may cause the column to clog thus resulting in decreased yield It is recommended to homogenize the tissue sample with rotor stator homogenizers since this method normally produces better yield Let sit at room temperature for 5 minutes Add 4 mL chloroform Vortex or shake vigorously for 15 seconds Let sit at room temperature for 2 3 minutes Centrifuge at 5 000 x g for 15 minutes at 4 C to separate the aqueous and organic phases Note The sample should separate into 3 phases an upper colorless aqueous phase which contains RNA a white inter phase and a lower blue organic phase Transfer the upper aqueous phase approximately 12 mL into a new 50 mL centrifuge tube not provided E Z N A Ultra Pure Total RNA Maxi Kit Protocols 8 Add an equal volume of 70 ethanol Vortex to mix thoroughly Note A precipitate may form at this point This will not interfere with the RNA purification 9 Insert a HiBind RNA Maxi Column into a 50 mL Collection Tube 10 Transfer 16 mL sample to the HiBind RNA Maxi Column 11 Centrifuge at 5 000 x g for 5 minutes 12 Discard the filtrate and reuse the collection tube 13 Repeat Steps 10 12 until all the
13. steps used are performed at room temperature unless otherwise noted Materials and Equipment to be Supplied by User Microcentrifuge capable of at least 5 000 x g and 4 C 70 ethanol in DEPC treated sterile distilled water Sterile RNase free pipette tips Nuclease free 50 mL centrifuge tubes 100 ethanol e Chloroform Homogenization Equipment Liquid nitrogen e Needle and syringe e Mortar and pestle Glass beads Rotor stator homogenizer Before Starting Prepare RNA Wash Buffer II according to the Preparing Reagents section on Page 5 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 Maxi Column The maximum amount of tissue that can be processed with is dependent on tissue type and its RNA content The maximum binding capacity of the HiBind RNA Maxi Column is 5 mg The maximum amount of tissue that can be used with RNA Solv Reagent is 1 g or 2 g adipose tissue 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 500 mg Based on RNA yield and quality obtained from 500 mg the starting amount can be adjusted for the next purification 10 E Z N A Ultra Pure Total RNA Maxi Kit Protocols Average Yield of Total Cellular RNA From Various Mouse Ti

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