Mag-Bind®Blood RNA 96 Kit - Omega Bio-Tek
Contents
1. 32 33 16 M2839 Mag Bind Blood RNA 96 Protocol Transfer the cleared supernatant containing purified RNA into a new RNase free microplate 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 beads 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 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 Incomplete resuspension Resuspend the magnetic particles by of magnetic particles vortexing before use Make sure samples are stored properly RNA degraded during and that the samples are processed sample storage immediately after collection or removal from storage Low RNA Yield RNA Wash Buffer II was Prepare RNA Wash Buffer II by adding not prepared correctly ethanol according to the instructions ie oft magnetic b ads Increase the bead collection time during procedure Blood clots cause congregation of magnetic beads Make sure the sample is clear of blood clots before adding magnetic beads RNA Wash Buffer II was No RNA eluted not diluted with 100 ethanol RNA is already degraded always use fresh Problem with blood for RNA2. Heat the DEPC Water to 70 C before adding to the beads 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 11 M2839 Mag Bind Blood RNA 96 Protocol Mag Bind Blood RNA 96 Kit Protocol M2839 200 uL blood Materials and Equipment to be Supplied by User Magnetic separation device for 96 well microplate Recommend Cat MSD 01 Nuclease free 1 mL or 2 mL 96 well deep well plate Recommend Cat EZ9602 01 Nuclease free 96 well microplate Recommend Cat EZ9604 01 Multichannel pipette Nuclease free pipette tips 100 ethanol Isopropanol Sealing film Before Starting 12 Prepare RNA Wash Buffer Il and RXT Wash Buffer according to the Preparing Reagents section on Page 6 Add 260 uL RBL Buffer and 260 uL isopropanol to each well of a 500 uL 96 well microplate Add 200 uL blood sample to each well and shake for 1 minute Add 20 uL Proteinase K Solution and 20 uL Mag Bind Particles CNR to each well Pipet up and down 10 times and shake for 5 minutes to mix thoroughly Note Proteinase K Solution must be added after the blood sample has been added to RBL Buffer Mag Bind Particles CNR and Proteinase K Solution can be made as a mastermix Let sit at room temperature for 10 minutes Place the plate on a magnetic separation device to magnetize the Mag Bind Particles CNR
3. Let sit at room temperature until the Mag Bind Particles CNR are completely cleared from solution Aspirate and discard the cleared supernatant Do not disturb the Mag Bind Particles CNR 10 11 12 13 14 15 16 M2839 Mag Bind Blood RNA 96 Protocol Remove the plate containing the Mag Bind Particles CNR from the magnetic separation device Add 600 uL RXT Wash Buffer to each well Pipet up and down 20 times or shake for 2 minutes to mix thoroughly Note RXT Buffer must be diluted with ethanol before use Please see Page 6 for instructions Place the plate on a magnetic separation device to magnetize the Mag Bind Particles CNR Let sit at room temperature until the Mag Bind Particles CNR are completely cleared from solution Aspirate and discard the cleared supernatant Do not disturb the Mag Bind Particles CNR Remove the plate containing the Mag Bind Particles CNR from the magnetic separation device Repeat Steps 8 11 for a second RXT Wash Buffer wash step Add 600 uL RNA Wash Buffer II to each well Pipet up and down 10 times or shake for 2 minutes to mix thoroughly Note RNA Wash Buffer II must be diluted with ethanol before use Please see Page 6 for instructions Place the plate on a magnetic separation device to magnetize the Mag Bind Particles CNR Let sit at room temperature until the Mag Bind Particles CNR are completely cleared from solution Aspirate and discard th
4. Mag Bind Particles CNR Let sit at room temperature until the Mag Bind Particles CNR are completely cleared from solution Aspirate and discard the cleared supernatant Do not disturb the Mag Bind Particles CNR Remove the plate containing the Mag Bind Particles CNR from the magnetic separation device Add 300 uL RNA Wash Buffer II to each well Pipet up and down 10 times or shake for 2 minutes to mix thoroughly Place the plate on a magnetic separation device to magnetize the Mag Bind Particles CNR Let sit at room temperature until the Mag Bind Particles CNR are completely cleared from solution Aspirate and discard the cleared supernatant Do not disturb the Mag Bind Particles CNR Leave the tube on the magnetic separation device for 10 minutes to air dry the Mag Bind Particles CNR Remove any residual liquid with a pipettor Add 20 50 uL DEPC Water Pipet up and down 20 times or shake for 1 minute to mix thoroughly Let sit at room temperature for 3 minutes Place the plate on a magnetic separation device to magnetize the Mag Bind Particles CNR Let sit at room temperature until the Mag Bind Particles CNR are completely cleared from solution 32 33 M2837 Mag Bind Blood RNA 96 Protocol Transfer the cleared supernatant containing purified RNA into a new RNase free microplate Store eluted RNA at 70 C Note Any combination of the following steps can be used to help increase RNA yield
5. isolation Prepare RNA Wash Buffer II by adding ethanol according to the instructions downstream Insufficient RNA was used applications Quantify the purified RNA accurately and use sufficient RNA Carryover Carryover of magnetic To remove the carryover magnetic beads of magnetic beads in the eluted from eluted RNA simply magnetize the beads during RNA will not effect magnetic beads and carefully transfer to elution downstream applications anew plate Make sure to use proper starting material Inefficient DNase aa contamination digestion Ensure that the DNase digestion is carried out at room temperature 17 18 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
6. scaled up or down If using the Mag Bind Blood RNA 96 Kit for the first time please read this booklet to become familiar with the procedure and its various modifications Samples are lysed in a specially formulated buffer containing detergent and chaotropic salt After adjusting the buffer conditions nucleic acids DNA RNA will form a complex with magnetic beads The beads nucleic acids complex is separated from lysates using a magnet Proteins and cellular debris are efficiently washed away by a washing step Next DNA is removed with a Mag Bind DNase treatment RNA is rebound and cleaned from the Mag Bind DNase reaction mixture using a second magnetic bead binding and washing procedure Pure RNA is eluted in nuclease free water or low ionic strength buffer Purified RNA can be directly used in downstream applications without the need for further purification New in this Edition This manual has been edited for content and redesigned to enhance user readability e DNase I has been replaced by Mag Bind DNase I This is a name change only Proteinase K is now supplied in a liquid form eliminating the resuspension step prior to use Proteinase K Solution can also be stored at room temperature for 12 months e Proteinase Storage Buffer is no longer included in the kit Before Beginning Important Notes Please take a few minutes to read this booklet in its entirety to become familiar with the procedures Prepare all materia
7. Ca OMEGA Innovations in nucleic acid isolation Product Manual Mag Bind Blood RNA 96 Kit 50 uL Blood M2837 00 1 x 96 preps M2837 01 4x 96 preps 200 uL Blood M2839 00 1 x 96 preps M2839 01 4 x 96 preps December 2013 For research use only Not intended for diagnostic testing Mag Bind Blood RNA 96 Kit Table of Contents Introduction and OVEFVIEW csssessescsecssseseecssecneesseecseeeseensees 2 Before BeginNniNg sssssesseessessesssssssssssssssssnseesenseesenneensstesteseeeeeee 3 Quantification of RNA mesrinrannsimeinaueiaaisa 4 Kit Contents Storage and Stability s sssssssssesesssssssssssssssssssse 5 Preparing Reagents essseesssserssseerssseessseressseessessossssssssseesssesssss 6 50 uL Mag Bind Blood RNA 96 Protocol M2837 7 200 uL Mag Bind Blood RNA 96 Protocol M2839 12 Troubleshooting Guide ssesssssseeessssessssssceeseesssssssseeeserssesssss 17 OPIS TIN a eta a A A 18 Manual Revision December 2013 N OMEGA bio tek Innovations in nucleic acid isolation Introduction and Overview The Mag Bind Blood RNA 96 Kit is designed for rapid and reliable isolation of total and viral RNA from mammalian whole blood The Mag Bind Bead technology provides high quality RNA which is suitable for direct use in most downstream applications such as amplifications and enzymatic reactions These protocols can be easily adapted to an automated system and the procedure can be
8. ature M2837 01 112 mL M2839 01 280 mL M2837 Mag Bind Blood RNA 96 Protocol Mag Bind Blood RNA 96 Kit Protocol M2837 50 uL blood Materials and Equipment to be Supplied by User Magnetic separation device for 96 well microplates Recommend Cat MSD 01 Nuclease free 500 uL 96 well microplates Recommend Cat EZ9604 01 e Multichannel pipette Nuclease free pipette tips 100 ethanol e lsopropanol e Sealing film Before Starting Prepare RNA Wash Buffer Il and RXT Wash Buffer according to the Preparing Reagents section on Page 6 1 Add 65 uL RBL Buffer and 65 uL isopropanol to each well of a 500 uL 96 well microplate 2 Add 50 uL blood sample to each well and shake for 1 minute 3 Add 5 ul Proteinase K Solution and 5 uL Mag Bind Particles CNR to each well Pipet up and down 10 times and shake for 5 minutes to mix thoroughly Note Proteinase K Solution must be added after the blood sample has been added to RBL Buffer Mag Bind Particles CNR and Proteinase K Solution can be made as a mastermix 4 Let sit at room temperature for 10 minutes 5 Place the plate on a magnetic separation device to magnetize the Mag Bind Particles CNR Let sit at room temperature until the Mag Bind Particles CNR are completely cleared from solution 6 Aspirate and discard the cleared supernatant Do not disturb the Mag Bind Particles CNR 10 11 12 13 14 15 16 M2837 Mag B
9. e cleared supernatant Do not disturb the Mag Bind Particles CNR Leave the tube on the magnetic separation device for 3 minutes to air dry the Mag Bind Particles CNR Remove any residual liquid with a pipettor 13 M2839 Mag Bind Blood RNA 96 Protocol 17 Prepare the Mag Bind DNase digestion mix as detailed in the table below Note If total nucleic acid DNA and RNA is desired skip Mag Bind DNase digestion steps Steps 18 24 and proceed to Step 25 for isolating both DNA and RNA Bi Numberor Mag Bind DNase Mag Bind DNase Total Volume Samples Digestion Buffer 96 1035m 211 uL 10 56 mL Volumes are calculated 10 extra to offset pipetting error Important Notes Mag Bind DNase is very sensitive and prone to physical denaturation Do not vortex the Mag Bind DNase I mixture Mix gently by shaking the plate Freshly prepare Mag Bind DNase digestion mix right before RNA isolation All steps must be carried out at room temperature Work quickly but carefully 18 Add 100 uL Mag Bind DNase digestion mix Pipet up and down 20 times or shake gently for 2 minutes to mix Note It is very important to remove any liquid drop from the wells before adding the Mag Bind DNase digestion mix Mag Bind DNase digestion mix must be used immediately once it is prepared 19 Let sit at room temperature for 10 15 minutes 20 Add 400 uL RNA Wash Buffer II Pipet up and down 20 time
10. ind Blood RNA 96 Protocol Remove the plate containing the Mag Bind Particles CNR from the magnetic separation device Add 200 uL RXT Wash Buffer to each well Pipet up and down 20 times or shake for 2 minutes to mix thoroughly Note RXT Buffer must be diluted with ethanol before use Please see Page 6 for instructions Place the plate on a magnetic separation device to magnetize the Mag Bind Particles CNR Let sit at room temperature until the Mag Bind Particles CNR are completely cleared from solution Aspirate and discard the cleared supernatant Do not disturb the Mag Bind Particles CNR Remove the plate containing the Mag Bind Particles CNR from the magnetic separation device Repeat Steps 8 11 for a second RXT Wash Buffer wash step Add 200 uL RNA Wash Buffer II to each well Pipet up and down 10 times or shake for 2 minutes to mix thoroughly Note RNA Wash Buffer II must be diluted with ethanol before use Please see Page 6 for instructions Place the plate on a magnetic separation device to magnetize the Mag Bind Particles CNR Let sit at room temperature until the Mag Bind Particles CNR are completely cleared from solution Aspirate and discard the cleared supernatant Do not disturb the Mag Bind Particles CNR Leave the tube on the magnetic separation device for 3 minutes to air dry the Mag Bind Particles CNR Remove any residual liquid with a pipettor M2837 Mag Bind Blood RNA 96 Protoc
11. ls 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 and plastic ware for 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 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 asses
12. ol 17 Prepare the Mag Bind DNase digestion mix as detailed in the table below Note If total nucleic acid DNA and RNA is desired skip Mag Bind DNase digestion steps Steps 18 24 and proceed to Step 25 for isolating both DNA and RNA i Numperof Mag Bing DNase Mag Bind DNase I Total Volume Samples Digestion Buffer ooo 9 58m 106 pL 5 29 mL Volumes are calculated 10 extra to offset pipetting error Important Notes Mag Bind DNase is very sensitive and prone to physical denaturation Do not vortex the Mag Bind DNase mixture Mix gently by shaking the plate Freshly prepare Mag Bind DNase digestion mix right before RNA isolation All steps must be carried out at room temperature Work quickly but carefully 18 Add 50 uL Mag Bind DNase digestion mix Pipet up and down 20 times or shake gently for 2 minutes to mix Note It is very important to remove any liquid drop from the wells before adding the Mag Bind DNase digestion mix Mag Bind DNase digestion mix must be used immediately once it is prepared 19 Let sit at room temperature for 10 15 minutes 20 Add 200 uL RNA Wash Buffer II Pipet up and down 20 times or shake for 5 minutes to mix 21 Let sit at room temperature for 5 minutes 22 23 24 25 26 27 28 29 30 31 10 M2837 Mag Bind Blood RNA 96 Protocol Place the plate on a magnetic separation device to magnetize the
13. s or shake for 5 minutes to mix 21 Let sit at room temperature for 5 minutes 14 22 23 24 25 26 27 28 29 30 31 M2839 Mag Bind Blood RNA 96 Protocol Place the plate on a magnetic separation device to magnetize the Mag Bind Particles CNR Let sit at room temperature until the Mag Bind Particles CNR are completely cleared from solution Aspirate and discard the cleared supernatant Do not disturb the Mag Bind Particles CNR Remove the plate containing the Mag Bind Particles CNR from the magnetic separation device Add 450 uL RNA Wash Buffer II to each well Pipet up and down 20 times or shake for 2 minutes to mix thoroughly Place the plate on a magnetic separation device to magnetize the Mag Bind Particles CNR Let sit at room temperature until the Mag Bind Particles CNR are completely cleared from solution Aspirate and discard the cleared supernatant Do not disturb the Mag Bind Particles CNR Leave the tube on the magnetic separation device for 10 minutes to air dry the Mag Bind Particles CNR Remove any residual liquid with a pipettor Add 100 uL DEPC Water Pipet up and down 20 times or shake for 1 minute to mix thoroughly Let sit at room temperature for 3 minutes Place the plate on a magnetic separation device to magnetize the Mag Bind Particles CNR Let sit at room temperature until the Mag Bind Particles CNR are completely cleared from solution 15
14. sed 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 Kit Contents Storage and Stability All Mag Bind Blood RNA 96 Kit components are guaranteed for at least 12 months from the date of purchase when stored as recommended Proteinase K Solution can be stored at room temperature for 12 months For long term storage gt 12 months store at 2 8 C Mag Bind Particle CNR must be stored at 2 8 C Mag Bind DNase must be stored at 20 C Store all other components at room temperature 22 25 C Check buffers for precipitates before use Redissolve any precipitates by warming to 37 C Preparing Reagents 1 Dilute RNA Wash Buffer II with 100 ethanol as follows and store at room temperature M2837 00 100 mL M2837 01 400 mL M2839 00 200 mL M2839 01 560 mL 2 Dilute RXT Wash Buffer with 100 ethanol as follows and store at room temper
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