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Manual - Omega Bio-Tek

1. 13 14 15 18 E Z N A Blood RNA Kit Protocols Add 250 uL RWF Buffer to the HiBind RNA Mini Column Centrifuge at 10 000 x g for 60 seconds 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 Incubate at room temperature for 15 minutes Add 250 uL RWF Buffer to the HiBind RNA Mini Column Incubate at room temperature for 2 minutes Centrifuge at 10 000 x g for 60 seconds Discard the filtrate and reuse the Collection Tube Add 700 uL RNA Wash Buffer II Note RNA Wash Buffer Il must be diluted with ethanol before use Please see Page 5 for instructions Centrifuge at 10 000 x g for 60 seconds Discard the filtrate and reuse the Collection Tube Repeat Steps 12 14 for a second RNA Wash Buffer II wash step 16 17 18 19 20 E Z N A Blood RNA Kit Protocols 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 t
2. 5 10 times 2 Proceed to Step 1 of the Protocol for Isolation of Total RNA from Animal Tissue on Page 13 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 12 E Z N A Blood RNA Kit Protocols 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 Protocol for Isolation of Total RNA from Animal Tissue 1 Determine the proper amount of starting material Note It is critical to use the correct amount of starting tissue to obtain optimal yield and purity with the HiBind RNA Mini Column The maximum amount of tissue that can be processed is dependent on the type of tissue and its RNA content The maximum binding capacity of the HiBind RNA Mini Column is 100 ug The maximum amount of tissue that can be used with 700 uL NTL Lysis Buffer is 30 mg Use the following table as a guide to select the correct amount of starting
3. Discard the filtrate and reuse the Collection Tube 16 Add 700 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 4 for instructions 17 Centrifuge at 10 000 x g for 30 seconds 18 Discard the filtrate and reuse the Collection Tube 15 19 20 21 22 23 16 E Z N A Blood RNA Kit Protocols Repeat Steps 16 18 for a second RNA Wash Buffer II wash step Centrifuge at maximum speed 212 000 x g 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 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 Preheat the DEPC Water to 70 C before adding to the column After adding the DEPC Water incubate the column for 5 minutes 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 main
4. E Z N A Blood RNA Kit Protocols 11 Add an equal volume of 70 ethanol Vortex to mix A precipitate may form after the addition of ethanol This will not interfere with RNA isolation 12 Insert a HiBind RNA Mini Column into a 2 mL Collection Tube provided with this kit 13 Transfer 700 uL including any precipitate to the HiBind RNA Mini Column 14 Centrifuge at gt 10 000 x g for 30 seconds 15 Discard the filtrate and reuse the Collection Tube 16 Repeat Steps 13 15 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 generally 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 17 See DNase Digestion Set Cat E1091 for further information If DNase digestion is not required proceed to Step 17 17 Add 500 uL RWF Wash Buffer to the HiBind RNA Mini Column 18 Centrifuge at gt 10 000 x g for 30 seconds 19 Discard the filtrate and the Collection Tube 20 Transfer the HiBind RNA Mini Column into a 2 mL Collection Tube provided with this kit 21 Add 700 uL RNA Wash Buffer Il Note RNA Wash Buffer Il must be diluted with ethanol befo
5. PCR use intron spanning primers that allow easy identification of DNA contamination A control PCR reaction containing the RNA as template will also allow detection of DNA contamination 10 E Z N A Blood RNA Kit Protocols E Z N A Blood RNA Kit Isolation of Total RNA from Animal Tissue Materials and Reagents to be Supplied by User 2 mercaptoethanol 70 ethanol diluted in nuclease free water RNase free pipette tips RNase free 1 5 mL microcentrifuge tubes RNase free 15 mL conical tubes depending on sample size Microcentrifuge capable of 13 000 x g Refrigerated microcentrifuge if available Centrifuge with swinging bucket rotor for 15 mL centrifuge tubes Before Starting Refrigerated microcentrifuge should be set to 4 C Prepare an ice bucket Prepare the following buffers 1 ERL Buffer and the RNA Wash Buffer Il according to the instructions on Page 4 2 NTL Lysis Buffer according to the instructions on Page 5 Sample Disruption and Homogenization Equipment Liquid nitrogen Omega Homogenizer Spin Columns Cat HCR001 HCROO3 Capless 2 0 mL Collection Tubes Cat SSI 1370 00 Needle and syringe Mortar and pestle Glass beads Rotor stator homogenizer 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
6. 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 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 E Z N A Blood RNA Kit Protocols E Z N A Blood RNA Kit Isolation of Total RNA from Blood Equipment and Reagents to be Supplied by User 2 mercaptoethanol 70 ethanol diluted in nuclease free water RNase free pipette tips RNase free 1 5 mL microcentrifuge tubes RNase free 15 mL conical tubes depending on sample size Microcentrifuge capable of 13 000 x g Refrigerated microcentrifuge if available Centrifug
7. 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 Mini Column to clog resulting in low or no yield 11 E Z N A Blood RNA Kit Protocols Liquid Nitrogen Method 1 Wear appropriate gloves and take great care when working with liquid nitrogen Excise tissue and promptly freeze in a small volume of liquid nitrogen Grind tissue with a ceramic mortar and pestle under approximately 10 mL liquid nitrogen 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 Allow the liquid nitrogen to completely evaporate and add NTL Lysis Buffer Proceed to one of the homogenization steps below Homogenization Choose one method below Homogenizer Spin Columns and 2 mL Collection Tubes Cat HCRO01 HCROO3 and SSI 1370 00 1 Load the lysate into a homogenizer spin column pre inserted into a2 mL Collection Tube 2 Centrifuge at maximum speed for 2 minutes in a microcentrifuge and save the filtrate 3 Proceed to Step 1 of the Protocol for Isolation of Total RNA from Animal Tissue on Page 13 Syringe and needle 1 Shear high molecular weight DNA by passing the lysate through a narrow needle 19 21 gauge
8. E Z N A Blood RNA Kit Table of Contents Introduction and OVEFVIEW csccsesssscsecssecseecseecseecseecneceseersees 2 Kit Contents Storage and Stability 3 Preparing Reagan iii 4 Before BedinniNd aus 5 Blood Proton un une 7 Tissue Poll 11 DNase Digestion Protocol e cocos 17 Troubleshooting Guide usassssessessesseeensenneeneensennenneensenneenee 20 OPS TING ooo 21 Manual Revision September 2011 Fi OMEGA bio tek Innovations in nucleic acid isolation Introduction and Overview Introduction E Z N A Blood RNA Kits are designed for isolation of total intracellular RNA from up to 1 mL of fresh whole blood treated with any common anticoagulant such as heparin EDTA or acid citrate dextrose Total RNA yield from 1 mL of blood ranges from 1 5 ug The Blood RNA Kit procedure efficiently removes contaminants and enzyme inhibitors making total RNA isolation fast convenient and reliable 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 This kit also is suitable for isolation of total RNA from cultured cells tissues bacteria and RNA viruses RNA purified using the E Z N A Blood RNA method is ready for applications such as RT PCR Overview The E Z N A Blood RNA Kits use the reversible binding properties of HiBind matrix a new silica based material This is combined w
9. Mini Column is approximately 100 ug the maximum amount of starting material depends on the lysis volume Due to the abundance of erythrocytes and proteins greater than 1 mL whole blood will significantly lower RNA quality Leukocytes have relatively low RNA content therefore the maximum binding capacity of HiBind RNA Mini Columns cannot be reached Samples should be collected in the presence of an anticoagulant preferably acid citrate dextrose and processed within a few hours Minimize storage time prior to RNA isolation as leukocyte transcripts generally have variable stabilities and avoid freezing the blood samples The E Z N A Blood RNA procedure involves erythrocyte lysis and removal which cannot be accomplished with frozen blood For such samples we recommend the modified protocol see Page 7 Note that only 150 uL frozen blood can be used with the modified procedure Before Beginning 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 treated 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 Ag ratio of 0 6 denotes pure protein A ratio of 1 8 2 0 corresponds to 90 100 pure nucleic acid
10. e with swinging bucket rotor for 15 mL centrifuge tubes Before Starting Refrigerated microcentrifuge should be set to 4 C Prepare an ice bucket Prepare the following buffers 1 ERL Buffer and the RNA Wash Buffer Il according to the instructions on Page 4 2 NTL Lysis Buffer according to the instructions on Page 5 Note After red blood lysis and removal all remaining steps must be carried out at room temperature Work quickly but carefully 1 Add 5 volumes ERL Buffer in an appropriately sized tube not provided Vortex to mix thoroughly For example add 5 mL ERL Buffer to 1 mL blood in a 15 mL conical tube or add 500 uL ERL Buffer to 100 uL blood in a 1 5 mL microcentrifuge tube Note ERL Buffer is supplied as a 10X concentrate and must be diluted with sterile distilled water before use Refer to Page 4 or the label on the bottle for directions 2 Incubate on ice for 15 minutes Vortex twice during incubation Note Lysis of red blood cells is indicated when the solution becomes translucent For blood samples from individuals with an elevated hematocrit or elevated erythrocyte sedimentation rate ESR extend the incubation time to 20 minutes 10 E Z N A Blood RNA Kit Protocols Centrifuge at 400 x g at 4 C for 10 minutes to pellet leukocytes Completely remove and discard the supernatant If a refrigerated centrifuge is not available centrifuge at room temperature but quickly complete Step 4 below Add 2 volu
11. ents 1 Dilute ERL Buffer with sterile distilled water as follows and store at room temperature P Kit OS Sterile Distilled Water to be Added R6814 01 450 mL R6814 02 450 mL per bottle 2 Dilute RNA Wash Buffer II with 100 ethanol as follows and store at room temperature R6814 01 100 mL R6814 02 200 mL Before Beginning Important Notes Please take a few minutes to read this booklet in its entirety to become familiar with the procedures 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 Prepare all materials required before starting the procedure to minimize RNA degradation e Carefully apply the sample or solution to the center of the HiBind RNA Mini Columns Avoid touching the membrane with pipet tips 2 mercaptoethanol is key in denaturing RNases and must be added to an aliquot of NTL Lysis Buffer before use Add 20 uL 2 mercaptoethanol per 1 mL NTL Lysis Buffer This mixture can be stored for 2 weeks at room temperature Starting Materials E Z N A Blood RNA Kits are designed for purification of total RNA from up to 1 mL fresh whole blood Although the binding capacity for the HiBind RNA
12. ith the speed of mini spin column technology Red blood cells are selectively lysed and white cells collected by centrifugation After lysis of white blood cells under denaturing conditions that inactivate RNases total RNA is purified with the HiBind spin column A specially formulated high salt buffer system allows RNA molecules greater than 200 bases to bind to the matrix Cellular debris and other contaminants such as hemoglobin are effectively washed away and high quality RNA is eluted in DEPC treated sterile water New in this Edition This manual has been edited for content and redesigned to enhance user readability Kit Contents Purifications HiBind RNA Mini Columns Homogenizer Columns 2 mL Collection Tubes ERL Buffer 10X NTL Lysis Buffer RWF Wash Buffer RNA Wash Buffer II DEPC Water User Manual Storage and Stability E Z N A Blood RNA Kits should be stored at room temperature During shipment crystals may form in the NTL Lysis Buffer Warm to 37 C to dissolve All kit components are guaranteed for 12 months from the date of purchase Modified Protocols E Z N A Blood RNA Kits may also be used for isolation of total RNA from cultured cells tissues bacteria and from acellular body fluids In addition RNA from enzymatic reactions such as in vitro transcription can be purified with this product For other modified protocols please contact our technical support or customer service Preparing Reag
13. material If information regarding the RNA content of your starting material is not available use 10 mg tissue Based on yield and quality of RNA obtained the starting amount can be adjusted up or down for the next purification Average Yield of Total Cellular RNA From Mouse Tissue nas 0 13 14 E Z N A Blood RNA Kit Protocols Add the appropriate amount of NTL Lysis Buffer based on the table below Amount of Tissue mg Amount of NTL Lysis Buffer uL 15 30 700 Note 2 mercaptoethanol is crucial for inactivating endogenous RNases and must be added to an aliquot of NTL Lysis Buffer Add 20 uL 2 mercaptoethanol per 1 mL NTL Lysis Buffer This mixture is stable at room temperature for 2 weeks Disrupt and homogenize the tissue sample with NTL Lysis Buffer Do not use more than 30 mg of tissue Homogenize cells with a rotor stator homogenizer or until the sample is uniformly homogenized Alternatively sample can be homogenized by other methods as described on Page 11 Note Incomplete homogenization of the sample will cause lower yields and clogging of the column It is recommended to homogenize the sample with rotor stator homogenizers since it normally produces better yields Centrifuge at 13 000 x g for 5 minutes Transfer the cleared supernatant to a clean 1 5 ml centrifuge tube not supplied Note In some preparations a fatty upper layer will form after centrifugation Transferring any of the fatty up
14. mes of ERL Buffer per volume of whole blood used in Step 1 Vortex to resuspend cells Note For example if you began the protocol with 1 mL whole blood wash the leukocyte pellet with 2 mL ERL Buffer Centrifuge at 400 x g at 4 C for 10 minutes Completely remove and discard the supernatant If a refrigerated centrifuge is not available centrifuge at room temperature but quickly complete Step 6 below Add NTL Lysis Buffer to the pelleted white blood cells For lt 500 uL whole blood add 400 uL NTL Lysis Buffer For 0 5 1 0 mL whole blood add 700 uL NTL Lysis Buffer Vortex to mix thoroughly Samples may be safely stored at 70 C after addition of NTL Lysis Buffer Note 2 mercaptoethanol is crucial for inactivating endogenous RNases and must be added to an aliquot of NTL Lysis Buffer Add 20 uL 2 mercaptoethanol per 1 mL NTL Lysis Buffer This mixture is stable at room temperature for 2 weeks Insert a Homogenizer Column into a 2 mL Collection Tube provided with this kit Transfer the cell lysate directly to the Homogenizer Column Note If too many cells have been used the cell lysate will be too viscous to pipet In this case divide the sample into two aliquots and adjust the volume of each aliquot to 700 uL with NTL Lysis Buffer Continue the protocol from Step 7 with two Homogenizer Columns and two HiBind RNA Mini Columns Centrifuge at maximum speed for 2 minutes Save the filtrate and discard the Homogenizer Column
15. o add water directly onto the HiBind RNA Mini Column matrix Incubate at room temperature for 1 minute Centrifuge at maximum speed for 2 minute 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 After adding the DEPC Water incubate the column for 5 minutes 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 19 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 Repeat elution Pre heat DEPC Water to 70 C prior to elution Incubate column for 10 minutes with water prior to centrifugation Reduce the quantity of the startin Column is overloaded q y 9 material Completely homogenize sample Increase centrifugation time Reduce the amount of starting material RNA remains on the Little orno column RNA eluted Incomplete homogenization Freeze starting material quickly in liquid nitrogen Do not store cultured cells prior to extraction unless they are lysed first Degraded RNA Follow protocol closely and work Source quickl
16. per layer may reduce RNA yields or clog the column Add an equal volume of 70 ethanol to the lysate Vortex to mix thoroughly Do not centrifuge If any sample has lost its volume during homogenization adjust the volume of ethanol accordingly Note A precipitate may form after the addition of ethanol in certain preparations This does not affect the procedure Insert a HiBind RNA Mini Column into a 2 mL Collection Tube provided with this kit E Z N A Blood RNA Kit Protocols 9 Transfer 700 uL of the sample including any precipitate that may have formed to the HiBind RNA Mini Column 10 Centrifuge at gt 10 000 x g for 30 seconds 11 Discard the filtrate and reuse the Collection Tube 12 Repeat Steps 9 11 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 generally 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 17 See DNase Digestion Set Cat E1091 for further information If DNase digestion is not required proceed to Step 13 13 Add 500 uL RWF Wash Buffer to the HiBind RNA Mini Column 14 Centrifuge at 10 000 x g for 30 seconds 15
17. re use Please see Page 4 for instructions 22 23 24 25 26 27 28 E Z N A Blood RNA Kit Protocols Centrifuge at gt 10 000 x g for 30 seconds Discard the filtrate and reuse the Collection Tube Repeat Steps 21 23 for a second RNA Wash Buffer II wash step 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 Transfer the HiBind RNA Mini Column to a clean 1 5 mL microcentrifuge tube not provided Add 50 100 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 Preheat the DEPC Water to 70 C before adding to the column After adding the DEPC Water incubate the column for 5 minutes 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 Note No RNA extraction procedure can completely remove genomic DNA For very sensitive work we suggest that you treat the eluted RNA with RNase free DNase Also for RT
18. taining elution volume E Z N A Blood RNA Kit Protocols E Z N A Blood RNA Kit DNase I Digestion Protocol Since the HiBind matrix of the RNA Column eliminates most DNA DNase digestion generally 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 16 of the Blood Protocol Pages 7 9 or Steps 1 12 of the Animal Tissue Protocol Pages 11 15 proceed with the following protocol User Supplied Material DNase Digestion Set Cat 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 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 Mini Column containing the sample into a 2 mL Collection Tube provided with this kit 17 10 11 12
19. y Ensure not to introduce RNase during RNase contamination the procedure Check buffers for RNase contamination Ensure RNA Wash Buffer II been diluted with 4 volumes of 96 100 ethanol as Salt carry over during indicated on bottle elution RNA Wash Buffer II must be stored and used at room temperature Repeat wash with RNA Wash Buffer II Reduce the amount of starting material Digest with RNase free DNase and inactivate at 75 C for 5 minutes Problem in downstream applications DNA HiBind DNA column contamination is overloaded DEPC Water is acidic and can RNA diluted in acidic dramatically lower Abs values buffer or water Use TE buffer to dilute RNA prior to spectrophotometric analysis Low Abs ratios 20 Ordering Information The following components are available for purchase separately Call Toll Free at 1 800 832 8896 Product Size Part Number DNase Digestion Kit 1500 Units 50 preps E1091 ERL Buffer 250 mL PRO23 RNA Wash Buffer II 50 mL PRO31 DEPC Water 100 mL PRO32 DNase RNase free Microcentrifuge Tubes 500 pk 10 pk cs SSI 1210 00 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 21 Notes 22 Notes 23 Notes 24

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