Manual - Omega Bio-Tek
Contents
1. Resuspend the Mag Bind Particles CNR by shaking for 2 minutes Let sit at room temperature for 10 minutes Place the plate on the 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 Transfer the cleared supernatant containing purified DNA RNA to a clean plate Store 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 Possible Problems and Suggestions Incomplete Resuspension Thoroughly resuspend Mag Bind of Magnetic Particles Particles CNR before use RNA Degraded during Immediately process sample after storage collection or removal from storage Low yield SPR Wash Buffer not Prepare SPR Wash Buffer with the prepared correctly correct amount of ethanol Double the volume of Proteinase Inefficient cell lysis K added to the sample and extend incubation by 5 minutes SS CS RNA in the sample already degraded do not freeze and thaw the sample more than once or Problem with Insufficient RNA was used a at room temperature for too downstream Quantify the purified DNA RNA applications accurately and use sufficient DNA RNA Dry the Mag Bind Particles CNR Ethanol carry over completely before adding elution buffer Place the eluted samples on a magne2. Mag Bind Viral DNA RNA 96 Kit Table of Contents Introduction and OVEFVIEW cscssccsscsscsseccecssecnseessecneceneerses 2 Kit Contents Storage and Stability sscsscsseesecseeeneers 3 Preparing Reagents essssesssseessseeessseeesseeessscesssseoossesssesosssseossss 4 Mag Bind Viral DNA RNA Protocol 50 yuL sssssssssssessesssss 5 Mag Bind Viral DNA RNA Protocol 200 uL sssessssssssss 8 Troubleshooting Guide ssesssssseeessssessssssteessesssssssseeeserssssssss 11 Ordering staen ae na ane eni 12 Manual Revision June 2012 024 OMEGA bio tek Innovations in nucleic acid isolation Introduction and Overview Introduction The Mag Bind Viral DNA RNA Kit is designed for rapid and reliable isolation of total nucleic acid from whole blood serum plasma saliva and other body fluids The Mag Bind paramagnetic bead technology provides high quality RNA or DNA which is suitable for direct use in most downstream applications such as amplifications and enzymatic reactions This system can be easily adapted to automated systems or centrifugation systems The procedure can be scaled up or down allowing purification from various amounts of starting material Overview If using the Mag Bind Viral DNA RNA 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 Nucleic acid is
3. bound to the surface of Mag Bind magnetic particles under proper condition Proteins and cellular debris are efficiently washed with few wash steps Pure RNA and DNA is then eluted in nuclease free water or low ionic strength buffer Purified RNA or DNA 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 Proteinase K is now supplied in a liquid form eliminating the step to respuspend prior to use Proteinase K Solution can be stored at room temperature for 12 months Proteinase Storage Buffer is no longer included in the kit Kit Contents Product M6246 01 M6246 02 Preparations 1 x 96 preps 4x 96 preps Mag Bind Particles CNR ell taal 44 mL TNA Lysis Buffer 30 mL 110 mL VHB Buffer 22 mL 88 mL Carrier RNA 1mg 4x1mg Proteinase K Solution 40 mg mL 1 1 mL 4 4 mL SPR Wash Buffer 25 mL 100 mL Nuclease free Water 35 mL 150 mL User Manual v Storage and Stability All components of the Mag Bind Viral DNA RNA Kit are stable for 24 months from the date of purchase when stored properly Mag Bind Particles CNR should be stored at 2 8 C Proteinase K Solution can be stored at room temperature for 12 months For long term store gt 12 months store at 2 8 C Carrier RNA should be stored at 20 C after reconstitution All other components should be stored at room temperature During sh
4. d for adequate washing of the Mag Bind Particles Place the plate on the 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 supernatant Do not disturb the Mag Bind Particles CNR Remove the plate from the magnetic separation device Add 500 uL SPR Wash Buffer to each well Note SPR Wash Buffer must be diluted with ethanol prior to use Please see Page 4 for instructions Resuspend the Mag Bind Particles CNR by shaking for 1 minute Place the plate on the 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 supernatant Do not disturb the Mag Bind Particles CNR 17 18 19 20 21 22 23 24 10 Mag Bind Viral DNA RNA 96 Kit Protocol Repeat Steps 12 16 for a second SPR Wash Buffer wash step Leave the plate on the magnetic separation device for 10 minutes to air dry the Mag Bind Particles CNR Remove any residual liquid with a pipettor Remove the plate from the magnetic separation device Add 50 100 uL Nuclease free Water to each well Note Elution volume depends on plastic ware and magnetic separation device used The Mag Bind Particles CNR must be able to completely covered by the Nuclease free Water
5. inute If using frozen samples thaw at room temperature and mix well by shaking or pipetting up and down before proceeding to Step 4 Note If the sample is less than 50 uL bring the volume up to 50 uL with Nuclease free water 4 Add 5 uL Mag Bind Particles CNR and 5 uL Proteinase K Solution to each well Mix by shaking for 5 minutes 5 Place the plate on a magnetic separation device to magnetize the Mag Bind Particles CNR Let sit for 10 15 minutes 10 11 12 13 14 15 16 17 Mag Bind Viral DNA RNA 96 Kit Protocol Aspirate and discard the supernatant Do not disturb the Mag Bind Particles CNR Remove the plate from the magnetic separation device Add 200 uL VHB Buffer to each well Note VHB Buffer must be diluted with ethanol prior to use Please see Page 4 for instructions Resuspend the Mag Bind Particles CNR by shaking for 1 minute Note Complete resuspension is required for adequate washing of the Mag Bind Particles Place the plate on the 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 supernatant Do not disturb the Mag Bind Particles CNR Remove the plate from the magnetic separation device Add 200 uL SPR Wash Buffer to each well Note SPR Wash Buffer must be diluted with ethanol prior to use Please see Page 4 for inst
6. ipment or storage in cool ambient conditions precipitates may form in the TNA Lysis Buffer It is possible to dissolve such deposits by warming the solution to 37 C We have determined that the precipitates do not interfere with overall performance Preparing Reagents Dilute VHB Buffer with 100 ethanol as follows as store at room temperature M6246 02 112 mL M6246 03 308 mL Dilute SPR Wash Buffer with 100 ethanol as follows as store at room temperature M6246 01 100 mL M6246 02 400 mL M6246 03 400 mL per bottle e Add Nuclease free Water to the tube containing lyophilized Carrier RNA to obtain a solution of 1 ug L Dissolve the carrier RNA thoroughly divide it into conveniently sized aliquots and store it at 20 C Do not freeze thaw the aliquots of Carrier RNA more than 3 times Mag Bind Viral DNA RNA 96 Kit Protocol Mag Bind Viral DNA RNA Kit Protocol 50 uL Sample Volume Materials and Equipment to be Supplied by User 100 Ethanol e lsopropanol Magnetic Separation Device for 96 well plates Cat MSD 01 96 well microplates U or V bottom Before Starting Prepare all Reagents according to Preparing Reagents section on Page 4 1 Freshly prepare the following lysis mastermix per sample me o a SE TNA Lysis Buffer 60 uL Isopropanol 70 uL 2 Transfer 132 uL lysis mastermix to each well of a 96 well microplate 3 Add 50 uL plasma or serum into each well Mix by shaking for 1 m
7. ment to be Supplied by User 100 Ethanol e lsopropanol Magnetic Separation Device for 96 well plates Cat MSD 01 96 well microplates U or V bottom Before Starting Prepare all Reagents according to Preparing Reagents section on Page 4 Freshly prepare the following lysis mastermix per sample ma e E TNA Lysis Buffer 240 uL Isopropanol 280 uL 2 Transfer 528 uL lysis mastermix to each well of a 96 well microplate 3 Add 200 uL plasma or serum into each well Mix by shaking for 1 minute If using frozen samples thaw at room temperature and mix well by shaking or pipetting up and down before proceeding to Step 4 Note If the sample is less than 200 uL bring the volume up to 200 uL with Nuclease free water 4 Add 10 uL Mag Bind Particles CNR and 10 uL Proteinase K Solution to each well Mix by shaking for 5 minutes 5 Place the plate on a magnetic separation device to magnetize the Mag Bind Particles CNR Let sit for 10 15 minutes 10 11 12 13 14 15 16 Mag Bind Viral DNA RNA 96 Kit Protocol Aspirate and discard the supernatant Do not disturb the Mag Bind Particles CNR Remove the plate from the magnetic separation device Add 400 uL VHB Buffer to each well Note VHB Buffer must be diluted with ethanol prior to use Please see Page 4 for instructions Resuspend the Mag Bind Particles CNR by shaking for 1 minute Note Complete resuspension is require
8. ructions Resuspend the Mag Bind Particles CNR by shaking for 1 minute Place the plate on the 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 supernatant Do not disturb the Mag Bind Particles CNR Repeat Steps 12 16 for a second SPR Wash Buffer wash step 18 19 20 21 22 23 24 Mag Bind Viral DNA RNA 96 Kit Protocol Leave the plate on the magnetic separation device for 10 minutes to air dry the Mag Bind Particles CNR Remove any residual liquid with a pipettor Remove the plate from the magnetic separation device Add 20 50 uL Nuclease free Water to each well Note Elution volume depends on plasticware and magnetic separation device used The Mag Bind Particles CNR must be able to completely covered by the Nuclease free Water Resuspend the Mag Bind Particles CNR by shaking for 2 minutes Let sit at room temperature for 10 minutes Place the plate on the 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 Transfer the cleared supernatant containing purified DNA RNA to a clean plate Store at 70 C Mag Bind Viral DNA RNA 96 Kit Protocol Mag Bind Viral DNA RNA Kit Protocol 200 uL Sample Volume Materials and Equip
9. tic separation device for an additional 5 minutes or centrifuge at gt 4 000 x g for 5 minutes Mag Bind Particles CNR would not fully magnetize on last step Carryover of Magnetic Beads 11 12 Ordering Information The following components are available for purchase separately Call Toll Free at 1 800 832 8896 E Z 96 Magnetic Separation Device for 96 well microplates MSD 01 500 uL Collection Plate 5 25 preps EZ9604 01 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
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