E.Z.N.A.®Viral RNA Kit - Omega Bio-Tek
Contents
1. Omega Bio tek s VAC 08 C Vacuum Tubing D Vacuum Source A Vacuum Manifold B Vacuum Flask E Z N A Viral RNA Kit Protocols E Z N A Viral RNA Kit Protocol Centrifugation Protocol All steps should be performed at room temperature Materials and Equipment to be Supplied by User Microcentrifuge capable of at least 13 000 x g 100 ethanol Sterile nuclease free 1 5 mL microcentrifuge tubes Sterile nuclease free pipette tips Before Starting Equilibrate samples and QVL Lysis Buffer to room temperature Prepare RNA Wash Buffer II VHB Buffer and Carrier RNA according to the Preparing Reagents section on Page 5 1 Prepare a master mix of QVL Lysis Buffer and Carrier RNA according to the table below Note QVL Lysis Buffer and Carrier RNA are stable at 2 8 C for 48 hours When stored at 2 8 C this mixture forms a precipitate that must be redissolved before use Warm the mixture to 80 C Do not warm for more than 5 minutes 10 11 12 13 14 15 E Z N A Viral RNA Kit Protocols Add 500 uL QVL Lysis Buffer into a 1 5 mL microcentrifuge tube not provided Add 150 uL plasma acellular body fluid cell culture supernatant or urine to the QVL Lysis Buffer Vortex for 30 seconds to mix thoroughly Let sit at room temperature for 5 10 minutes Centrifuge briefly to collect any liquid droplets from the lid Add 350 uL 100 ethanol Vortex for 30 seconds2. Ca OMEGA Innovations in nucleic acid isolation bio tek Product Manual E Z N A Viral RNA Kit R6874 00 5 preps R6874 01 50 preps R6874 02 200 preps September 2012 For research use only Not intended for diagnostic testing E Z N A Viral RNA Kit Table of Contents Introduction and OVEIMEW ccissstsennnnendnanaisinaninennes 2 Illustrated PrOtOCGlS ccseicsscisiccsecornenieadontamnnniiaencnnnn 3 Kit Contents Storage and Stability ssssssssecseecneersees 4 Preparing CACIST UES castes sonssysseysscutessestussssvsenibacesstvcestucsnsuseneteastenets 5 Recommended Steins scctscsssscscenserssrissanennnenensninannes 6 Centrifugation Protocol vacisnniacnicananumaumcianmmians 7 Vacu m PROLOCO i ecieccecisincisdeecinaennoaid ean 10 Troubleshooting Guide ssssssssseressssessssssteeseesssssssseeeserssesssss 13 POSTING eein AEE E EAE 14 Manual Revision September 2012 02 OMEGA bio tek Innovations in nucleic acid isolation Introduction and Overview E Z N A Viral RNA Kit is designed for isolation of viral RNA from cell free 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 tested for isolating viral nucleic acids from hepatitis A C and HIV The kit is also suitable for isolation of total RNA from cultured cells tissues and gram negative bacteria RNA pur
3. NA Kit components are guaranteed for at least 12 months from the date of purchase when stored as follows Carrier RNA must be stored at 20 C All other components can be stored at room temperature During shipment or storage in cool ambient conditions precipitates may form in QVL Lysis Buffer Dissolve such deposits by warming the solution at 37 C and gently shaking Preparing Reagents 1 Dilute RNA Wash Buffer II with 100 ethanol as follows and store at room temperature R6874 02 200 mL 2 Add DEPC Water to the tube of lyophilized Carrier RNA as follows Dissolve the Carrier RNA completely aliquot and store at 20 C Do not freeze thaw the aliquots more than three times ec a 3 Dilute VHB Buffer with 100 ethanol as follows and store at room temperature R6874 01 19 1 mL Recommended Settings The following is required for use with the Vacuum Protocol A Vacuum Manifold We recommend Omega Bio tek s VAC 08 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 Manifold Recommended Pressure mbar VAC 08 200 to 600 Conversion from millibars Multiply by Millimeters of mercury mm Hg 0 75 Inches of mercury inch Hg 0 0295 Tors Tor Atmospheres atmos 0 000987 Pounds per Square Inch psi 0 0145 Illustrated Vacuum Setup
4. VL Lysis Buffer Vortex for 30 seconds to mix thoroughly Let sit at room temperature for 5 10 minutes Centrifuge briefly to collect any liquid droplets from the lid Add 350 uL 100 ethanol Vortex for 30 seconds to mix thoroughly Centrifuge briefly to collect any liquid droplets from the lid Prepare the vacuum manifold according to manufacturer s instructions and connect the HiBind RNA Mini Column to the manifold Transfer 750 uL sample including any precipitate to the HiBind RNA Mini Column Switch on vacuum source to draw the sample through the column Note If for any reason the solution has trouble passing through the column turn off the vacuum transfer the column to a 2 mL Collection Tube centrifuge at maximum speed for 5 minutes or until all the sample passes through the column Continue with Step 9 of the Centrifugation Protocol on Page 8 Turn off the vacuum Repeat Steps 9 11 until all the lysate has been transferred to the HiBind RNA Mini Column Add 500 uL VHB Buffer Note VHB Buffer must be diluted with ethanol before use Please see the Preparing Reagents section on Page 5 for instructions 11 14 V5 16 17 18 19 20 21 22 23 24 25 12 E Z N A Viral RNA Kit Protocols Switch on vacuum source to draw the VHB Buffer through the column Turn off the vacuum Add 500 uL RNA Wash Buffer II Note RNA Wash Buffer II must be diluted with ethanol bef
5. ified using the E Z N A Viral RNA method is ready for applications such as RT PCR The E Z N A Viral RNA Kits use the reversible binding properties of HiBind matrix a silica based time saving material Combined with the speed of mini column spin technology or vacuum manifold multiple samples can be processed at the same time The sample is lysed under highly denaturing buffer conditions so that RNases are inactivated and the intact viral RNA is protected from degradation After adjusting the buffer conditions the samples are transferred to the HiBind RNA Mini Column With brief centrifugation or vacuum the samples pass through the column and the viral RNA binds to the HiBind matrix After two wash steps purified viral RNA is eluted with RNase free water E Z N A Viral RNA Kits are not designed to separate viral RNA from cellular RNA and DNA It will purify both in parallel if they present in the sample Acellular body fluids are recommended New in this Edition This manual has been edited for content and redesigned to enhance user readability Centrifugation Protocol Vacuum Protocol Add QVL Lysis Buffer and Lyse Add Ethanol Apply Sample i to Column 3 Wash 3X i Dry N RJ CN KD Elute Add QVL Lysis Buffer and Lyse Add Ethanol Apply Sample to Column Wash 3X Dry Elute Kit Contents fantcotecintwpes a oo Storage and Stability All of the E Z N A Viral R
6. le for purchase separately Call Toll Free at 1 800 832 8896 product Parr 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 14
7. lications Transfer the HiBind RNA Mini Column to a clean 1 5 mL microcentrifuge tube not provided Add 20 50 uL DEPC Water directly to the center of column matrix Centrifuge at maximum speed for 1 minute Store RNA at 70 C E Z N A Viral RNA Kit Protocols E Z N A Viral RNA Kit Protocol Vacuum Protocol All steps should be performed at room temperature Materials and Equipment to be Supplied by User e Vacuum manifold with standard luer adaptor Microcentrifuge capable of at least 13 000 x g 100 ethanol Sterile nuclease free 1 5 mL microcentrifuge tubes Sterile nuclease free pipette tips Before Starting Equilibrate samples and QVL Lysis Buffer to room temperature Prepare RNA Wash Buffer II VHB Buffer and Carrier RNA according to the Preparing Reagents section on Page 5 1 Prepare a master mix of QVL Lysis Buffer and Carrier RNA according to the table below Note QVL Lysis Buffer and Carrier RNA are stable at 2 8 C for 48 hours When stored at 2 8 C this mixture forms a precipitate that must be redissolved before use Warm the mixture to 80 C Do not warm for more than 5 minutes eid CS w Oo os J o d s Ek a 8 Oo o n o e 10 10 11 12 13 E Z N A Viral RNA Kit Protocols Add 500 uL QVL Lysis Buffer into a 1 5 mL microcentrifuge tube not provided Add 150 uL plasma acellular body fluid cell culture supernatant or urine to the Q
8. ore use Please see the Preparing Reagents section on Page 5 for instructions Switch on vacuum source to draw the RNA Wash Buffer II through the column Turn off the vacuum Repeat Steps 16 18 for a second RNA Wash Buffer II wash step Transfer the HiBind RNA Mini Column to a new 2 mL Collection Tube Centrifuge the empty HiBind RNA Mini Column at maximum speed for 2 minutes to dry the column Note This step is critical for removal of trace ethanol that may interfere with downstream applications Transfer the HiBind RNA Mini Column to a clean 1 5 mL microcentrifuge tube not provided Add 20 50 uL DEPC Water directly to the center of column matrix Centrifuge at maximum speed for 1 minute Store RNA at 70 C 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 Ensure RNA Wash Buffer II has been diluted with 4 volumes of 100 Salt carry over during ethanol as indicated on bottle elution RNA Wash Buffer II must be stored at dee room temperature applications Repeat wash with RNA Wash Buffer II Problem in downstream PCR Inhibitors Dilute the starting sample with PBS Buffer ae ee eee Perform an on membrane DNase DNA contamination digestion Refer to Product E1091 for more details contamination 13 Ordering Information The following components are availab
9. to mix thoroughly Centrifuge briefly to collect any liquid droplets from the lid Insert a HiBind RNA Mini Column into a 2 mL Collection Tube provided Transfer 750 uL sample including any precipitate to the HiBind RNA Mini Column Centrifuge at maximum speed 213 000 x g for 15 seconds Discard filtrate and reuse the collection tube Repeat Steps 9 11 until all the sample has been transferred to the HiBind RNA Mini Column Transfer the HiBind RNA Mini Column to a new 2 mL Collection Tube Add 500 uL VHB Buffer Note VHB Buffer must be diluted with ethanol before use Please see the Preparing Reagents section on Page 5 for instructions Centrifuge at maximum speed for 15 seconds 16 17 18 19 20 21 22 23 24 25 26 E Z N A Viral RNA Kit Protocols Discard the filtrate and the collection tube Transfer the HiBind RNA Mini Column to a new 2 mL Collection Tube Add 500 uL RNA Wash Buffer II Note RNA Wash Buffer II must be diluted with ethanol before use Please see the Preparing Reagents section on Page 5 for instructions Centrifuge at maximum speed for 15 seconds Discard filtrate and reuse the collection tube Repeat Steps 18 20 for a second RNA Wash Buffer Il wash step Centrifuge the empty HiBind RNA Mini Column at maximum speed for 2 minutes to dry the column Note This step is critical for removal of trace ethanol that may interfere with downstream app
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