Home

Manual

1. Materials and Equipment to be Supplied by User Vacuum Manifold Cat VAC 03 Vacuum source e Multichannel pipet RNase free filter tips Reagent reservoirs for multichannel pipet RNase free 1 2 mL 96 well plates Sealing film 100 ethanol Before Starting Prepare RNA Wash Buffer Il and QVL Lysis Buffer carrier RNA according to the Preparing Reagents section on Page 5 Equilibrate QVL Lysis Buffer carrier RNA to room temperature before use 1 Add 150 uL plasma acellular body fluid cell culture or urine to each well of a 1 2 mL 96 well plate not provided 2 Add 10 uL OB Protease Solution to each well 3 Add 500 uL QVL Lysis Buffer carrier RNA solution to each well Seal the plate the AeraSeal Film 4 Keeping the 96 well plate flat on the bench shake vigorously back and forth for 30 seconds Rotate the plate 90 and shake the plate for another 30 seconds 5 Let sit at room temperature for 10 minutes 6 Remove and discard the AeraSeal Film 11 10 11 12 13 14 15 16 17 18 19 12 E Z 96 Viral RNA Kit Protocols Add 350 uL 100 ethanol to each well Pipet up and down 4 times to mix thoroughly Assemble the vacuum manifold according to the manufacturer s instructions Transfer 500 uL sample from Step 6 including any precipitate that may have formed to each well of the E Z 96 RNA Plate Note Seal any unused wells with sealing film not AeraSeal Film
2. Turn on the vacuum source to draw the sample through the plate Turn off the vacuum Repeat Steps 8 10 until all the sample has been transferred to the E Z 96 RNA Plate Add 750 uL RWF Wash Buffer to each well Turn on the vacuum source to draw the RWF Wash Buffer through the plate Turn off the vacuum Lift the Vacuum Manifold Collar containing the E Z 96 RNA Plate from the Vacuum Manifold Base and empty the waste Reassemble the manifold Add 500 uL RNA Wash Buffer II to each well Note RNA Wash Buffer II must be diluted with 100 ethanol before use Please follow the instructions on Page 5 Turn on the vacuum source to draw the RNA Wash Buffer Il through the plate Turn off the vacuum 20 21 22 23 24 25 26 27 28 29 30 31 32 E Z 96 Viral RNA Kit Protocols Repeat Steps 16 18 for a second RNA Wash Buffer Il wash step Place the E Z 96 RNA Plate on a stack of paper towels and tap the bottom tip side several times to remove any residual ethanol Return the E Z 96 RNA Plate to the vacuum manifold Turn on the vacuum source for 15 minutes Turn off the vacuum Replace the Waste Tray with the 96 well Racked Microtubes provided and reassemble the manifold Add 50 70 uL DEPC Water to each well Seal the E Z 96 RNA Plate with new AeraSeal Film Make sure to add the DEPC Water directly to the center of each well Let sit for 1 minute at room temperature Turn on the va
3. Ca OMEGA Innovations in nucleic acid isolation Product Manual E Z 96 Viral RNA Kit R1074 00 1 x 96 preps R1074 01 4x 96 preps October 2013 For research use only Not intended for diagnostic testing E Z 96 Viral RNA Kit Table of Contents Introduction and OVErVIEW cesseecssecseessescscececssceneeeseecsecsees Kit Contents Storage and Stability ssessescssececsseenes Important NOTES ssscesssinssnsciesnscoveszonessesnveacasve eassdeceveasscvsnocesiesenviss Preparing Reagents s sssssesssssssseeesssssssessssssseesssssessssseeesssssseee Vacuum Manifold Information cccscssecsessscssessssseesneesees Centrifugation ProtocOl esssssssecsesseesscsscssscssecsseseesnsenceneeees Vacuum Protocolar nann Troubleshooting Guide ssesssssseeesessssssesseeeseesssssssseeeseessesssss Or a e E A AE Manual Revision October 2013 N OMEGA bio tek Innovations in nucleic acid isolation Introduction and Overview E Z 96 Viral RNA Kit is designed for isolation of viral RNA from acellular fluids such as plasma serum urine and cell culture supernatant The procedure completely removes contaminants and enzyme inhibitors making viral RNA isolation fast convenient and reliable This kit has been validated with hepatitis A and C and HIV The kit is also suitable for isolation of total RNA from cultured cells tissues and bacteria RNA purified using the E Z 96 Viral RNA method is ready for applications such as RT
4. PCR The E Z 96 Viral RNA Kit uses the reversible binding properties of the silica based HiBind matrix combined with the speed of mini column spin technology or vacuum manifold to process multiple samples quickly and efficiently The sample is lysed under denaturing conditions that inactivate RNases and protects the intact viral RNA from degradation After adjusting the binding conditions the samples are transferred to the E Z 96 RNA Plate With a brief centrifugation or vacuum step the samples pass through the plate and the viral RNA binds to the Hibind matrix After two wash steps purified viral RNA is eluted with RNase free water Note E Z 96 Viral RNA Kits are not designed to separate viral RNA from cellular RNA and DNA It will purify both if they are present in the sample Acellular body fluids are recommended Sample Volumes HiBind RNA matrix can bind any RNA greater than 200 nt Yield will depend on the sample source and condition The protocol is optimized for use with a sample volume of 150 uL Smaller samples should be adjusted to 150 uL with PBS or DEPC treated water Lower titer samples should be concentrated to 150 uL before processing For samples larger than 150 uL the amount of QVL Lysis Buffer and other reagents added to the sample before loading must be increased proportionally New in this Edition This manual has been edited for content and redesigned to enhance user readability OB Protease is now supplied
5. Seal Film Transfer the E Z 96 RNA Plate to a clean 2 mL 96 well plate not provided Add 500 uL RNA Wash Buffer II to each well Seal the plate with AeraSeal Film Note RNA Wash Buffer II must be diluted with 100 ethanol before use Please follow the instructions on Page 5 22 23 24 25 26 27 28 29 30 31 32 33 34 35 10 E Z 96 Viral RNA Kit Protocols Centrifuge at 4 000 x g for 5 minutes at room temperature Discard the filtrate from the 96 well plate Remove and reuse the AeraSeal Film in the following step Repeat Steps 23 26 for a second RNA Wash Buffer II wash step Centrifuge at 4 000 x g for 10 minutes at room temperature Note It is important to dry the E Z 96 RNA Plate matrix before elution Residual ethanol may interfere with downstream applications Remove and discard the AeraSeal Film Transfer the E Z 96 RNA Plate to the 96 well Racked Microtubes provided Add 50 70 uL DEPC Water to each well Seal the plate the new AeraSeal Film Make sure to add water directly onto RNA matrix Let sit for 1 minute at room temperature Centrifuge at 4 000 x g for 5 minutes at room temperature Remove and reuse the AeraSeal Film in the following step Repeat Steps 30 32 for a second elution step Seal the 96 well Racked Microtubes with Caps for Racked Microtubes Store RNA at 70 C E Z 96 Viral RNA Kit Protocols E Z 96 Viral RNA Kit Protocol Vacuum Protocol
6. cuum source for 5 minutes Turn off the vacuum Repeat Steps 25 28 for a second elution step Seal the 96 well Racked Microtubes with Caps for Racked Microtubes Store RNA at 70 C 13 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 Dissolve the carrier RNA with QVL Lysis Carrier RNA Buffer and repeat the purification with a not added to new sample QVL Buffer or Avoid warming the QVL carrier RNA degraded Repeat elution RNA remains Preheat DEPC Water to 70 C prior to elution on the plate Let sit for 5 minutes with DEPC Water prior to elution Plate is overloaded Reduce the amount of starting material Mix thoroughly after addition of QVL Lysis Clogged well Incomplete lysis Buffer Reduce the amount of the starting material Do not freeze thaw sample more than once Source Follow protocol closely and work quickly Low concentration of virus in the sample Degraded RNA Little or no RNA eluted Ensure not to introduce RNase during the procedure Check buffers for RNase contamination Ensure RNA Wash Buffer II has been diluted Salt carryover with 100 ethanol as instructed RNA Wash Buffer II must be stored at room Problem in during elution temperature downstream Repeat wash with RNA Wash Buffer Il applications RNase contamina tion Use less starting materia
7. fer carrier RNA solution to each well Seal the plate with AeraSeal Film 4 Keeping the 96 well plate flat on the bench shake vigorously back and forth for 30 seconds Rotate the plate 90 and shake the plate for another 30 seconds 5 Let sit at room temperature for 10 minutes 6 Briefly centrifuge at 500 x g to collect any liquid droplets from the film 7 Remove and discard the AeraSeal Film 10 11 12 13 14 15 16 17 18 19 20 21 E Z 96 Viral RNA Kit Protocols Add 350 uL 100 ethanol to each well Seal the plate with AeraSeal Film Vortex the plate for 30 seconds Briefly centrifuge at 500 x g to collect any liquid droplets from the film Remove and discard the AeraSeal Film Place an E Z 96 RNA Plate onto a 96 well Square well Plate 2 2 mL provided Transfer 500 uL sample from Step 8 including any precipitate that may have formed to each well of the E Z 96 RNA Plate Seal the E Z 96 RNA Plate with AeraSeal Film Centrifuge at 4 000 x g for 5 minutes at room temperature Discard the filtrate from the 96 well Square well Plate Remove and reuse the AeraSeal Film in the following step Repeat Steps 11 15 until all the sample has been transferred to the E Z 96 RNA Plate Remove and discard the AeraSeal Film Add 750 uL RWF Wash Buffer to each well Seal the plate with AeraSeal Film Centrifuge at 4 000 x g for 5 minutes at room temperature Remove and discard the Aera
8. gest that you dilute the sample in a buffered solution TE for spectrophotometric analysis The A A ratio of pure nucleic acids is 2 0 while an A A ratio of 0 6 denotes pure protein A ratio of 1 8 2 0 corresponds to 90 100 pure nucleic acid Phenol has a maximum absorbance at 270 nm and can interfere with spectrophotometric analysis of DNA or RNA Store RNA samples at 70 C in water Under these conditions RNA is stable for more than a year Integrity of RNA It is highly recommended that RNA quality be determined prior to beginning all downstream applications The quality of RNA can be best assessed by denaturing agarose gel electrophoresis with ethidium bromide staining The ribosomal RNA bands should appear as sharp clear bands on the gel The 28S band should appear to be double that of the 18S RNA band 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 Cleaning of 96 well Square well Plates The 96 well Square well Plates supplied with this kit are reusable To avoid cross contamination rinse the plates thoroughly with tap water after each u
9. in a liquid form eliminating the resuspension step prior to use OB Protease Solution can be stored at room temperature for 12 months Proteinase Storage Buffer is no longer included in the kit Kit Contents fase f The 96 well Square well Plates supplied with this kit are reusable Please refer to Page 4 for cleaning instructions Storage and Stability All of the E Z 96 Viral RNA Kit components are guaranteed for at least 12 months from the date of purchase when stored as follows QVL Lysis Buffer carrier RNA solution is stable for up to 6 months when stored at 2 8 C QVL carrier RNA solution is only stable for a maximum of 14 days when stored at room temperature It is recommended that aliquots of this buffer be made according to average usage per week OB Protease Solution can be stored at room temperature for up to 12 months For long term storage store OB Protease Solution at 2 8 C All remaining components should be stored at room temperature During shipment or storage in cool ambient conditions precipitates may form in the QVL Lysis Buffer Dissolve such deposits by warming the solution at 37 C and gently shaking Important Notes 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 sug
10. l Inhibitors of PCR Increase incubation with QVL Lysis Buffer to completely lyse cells DNA Digest with RNase free DNase and inactivate at contamination 75 C for 5 minutes 14 Ordering Information The following components are available for purchase separately Call Toll Free at 1 800 832 8896 C 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 15 Notes
11. se Soak the plates in 0 5M HCI for 5 minutes then wash thoroughly with distilled water The 96 well Square well Plates also can be autoclaved following washing Isolation of Cellular Bacterial or Viral DNA from Urine The QVL Lysis Buffer can inactivate numerous PCR inhibitors found in urine This product can be used for isolation of cellular bacterial or viral DNA from urine for use in PCR We recommend the use of the centrifugation protocol Since urine contains a very low number of cells bacteria and viruses samples often need to be concentrated to a final volume of 150 uL Preparing Reagents Dilute RNA Wash Buffer II with 100 ethanol as follows and store at room temperature R1074 00 200 mL R1074 01 200 mL Resuspension of carrier RNA with QVL Lysis Buffer 1 Add 1 mL QVL Lysis Buffer to the vial of carrier RNA 2 Shake the vial to completely dissolve the carrier RNA 3 Transfer the dissolved carrier RNA to the bottle of QVL Lysis Buffer 4 Store at 2 8 C Vacuum Manifold Information 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 millibars Mul
12. tiply 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 Vacuum Manifold Information RNA Bind amp Wash Setup _ E Z 96 RNA Plate Vacuum Manifold Collar Waste Collection 96 well Square well Plate Vacuum Manifold Base E Z 96 RNA Plate Vacuum Manifold Collar 96 well Microplate 500 uL Waste Collection Vacuum Manifold Base E Z 96 Viral RNA Kit Protocols E Z 96 Viral RNA Kit Protocol Centrifugation Protocol Materials and Equipment to be Supplied by User e Centrifuge capable 4 000 x g with adaptor for 96 well plates Vortexer e Multichannel pipet RNase free filter tips Reagent reservoirs for multichannel pipet RNase free 1 2 mL 96 well plates e 2 mL96 well plates 100 ethanol Before Starting Prepare RNA Wash Buffer II and QVL Lysis Buffer carrier RNA solution according to the Preparing Reagents section on Page 5 Equilibrate QVL Lysis Buffer carrier RNA to room temperature before use 1 Add 150 uL plasma acellular body fluid cell culture or urine to each well of a 1 2 mL 96 well plate not provided 2 Add 10 uL OB Protease Solution to each well 3 Add 500 uL QVL Lysis Buf

Manual

Contents

Download Pdf Manuals

Related Search

Related Contents