Mag-Bind®Circulating DNA Kit - Omega Bio-Tek
Contents
1. 18 19 20 21 22 23 24 25 26 27 Mag Bind Circulating DNA Kit Protocol Remove the plate containing the Mag Bind Particles RQ from the magnetic separation device Add 3 mL DNA Wash Buffer to each sample Note DNA Wash Buffer must be diluted with 100 ethanol prior to use Please see Page 4 for instructions Resuspend the Mag Bind Particles RQ by pipetting up and down 20 times or vortexing for 1 minute Let sit at room temperature for 1 minute Place the plate on the magnetic separation device to magnetize the Mag Bind Particles RQ Let sit at room temperature until the Mag Bind Particles RQ are completely cleared from solution Aspirate and discard the cleared supernatant Do not disturb the Mag Bind Particles RQ Repeat Steps 15 20 using 800 uL DNA Wash Buffer Remove the plate from the magnetic separation device for approximately 30 seconds Place the plate on the magnetic separation device to magnetize the Mag Bind Particles RQ Aspirate and discard the residual DNA Wash Buffer Leave the plate on thet magnetic separation device Add 500 uL water to each sample and immediately remove within 30 seconds Remove the plate containing the Mag Bind Particles RQ from the magnetic separation device 28 29 30 31 32 Mag Bind Circulating DNA Kit Protocol Add 200 400 uL Elution Buffer or nuclease free water to elute DNA from the Mag Bind Particles RQ Resuspend2. that will fit your needs Utilizing paramagnetic particles provides high quality DNA that is suitable for direct use in most downstream applications such as qPCR and Next Generation Sequencing Overview If using the Mag Bind Circulating DNA Kit for the first time please read this booklet in its entirety to become familiar with the procedures Blood cells are lysed in a specially formulated buffer DNA is isolated from the lysates in one step by binding to Mag Bind Particles surfaces The paramagnetic particles are separated from the lysates by using a magnetic separation device After a few rapid wash steps to remove trace contaminants DNA is eluted in Elution Buffer Kit Contents Storage and Stability All of the Mag Bind Circulating DNA Kit components are guaranteed for at least 12 months from the date of purchase when stored as follows Mag Bind Particles RQ should be stored at 2 8 C for long term use Proteinase K Solution can be stored at room temperature for up to 12 months For long term storage store Proteinase K Solution at 2 8 C Preparing Reagents 1 Dilute DNA Wash Buffer with 100 ethanol as follows and store at room temperature M3291 00 100 mL M3291 01 100 mL per bottle 2 Prepare VHB Buffer with 100 ethanol as follows and store at room temperature M3291 01 56 mL per bottle 3 Prepare ACB Buffer with 100 isopropanol as follows and store at room temperature M3291 01 240 mL 4 S
3. the Mag Bind Particles RQ by pipetting up and down or vortexing Let sit at room temperature for 5 minutes Place the plate on the magnetic separation device to magnetize the Mag Bind Particles RQ Let sit at room temperature until the Mag Bind Particles RQ are completely cleared from solution Transfer the cleared supernatant containing purified DNA to a clean microplate or 1 5 mL centrifuge tube not supplied Store DNA at 20 C Mag Bind Circulating DNA Kit Protocol Lia Circulating DNA Kit Protocol for 500 1000 uL Serum Plasma Materials and Reagents to be Supplied by User 100 ethanol e 100 isopropanol Magnetic separation device for 24 well plates Cat A000270 or Magnetic Separator for 15 mL centrifuge tubes e Vortexer 24 well plate 10 mL Cat Whatman 7701 5102 or 15 centrifuge tubes Incubator capable of 60 C 1 5 mL microcentrifuge tubes Optional PBS or nuclease free water Before Starting Prepare ACB Buffer DNA Wash Buffer and VHB Buffer according to the Preparing Reagents section on Page 4 Set Incubator to 60 C 1 Add 500 1000 uL plasma serum samples to a 10 mL 24 well plate not provided or 15 mL centrifuge tube not provided Choose the correct plasticware depending on magnetic stand being utilized to process the samples Bring the volume up to 1 mL with Elution Buffer provided with this kit if volume of sample is less than 1 mL 2 Add 100 uL Proteinase
4. Ca OMEGA Innovations in nucleic acid isolation bio tek Product Manual Mag Bind Circulating DNA Kit M3291 00 1 x 24 preps M3291 01 4 x 24 preps October 2014 For research use only Not intended for diagnostic testing Mag Bind Circulating DNA Kit Table of Contents Introduction and Overview 2 Kit Contents Storage and StabiliW un nennen 3 Preparing Reagents nenne 4 Mag Bind Circulating DNA Protocol for 1000 2000 pL 5 Mag Bind Circulating DNA Protocol for 500 1000 wL 9 Troubleshooting Gilde atacada 13 Ora see 14 Manual Revision October 2014 Fi OMEGA bio tek Innovations in nucleic acid isolation Introduction and Overview Introduction The Mag Bind Circulating DNA Kit is designed for rapid and reliable isolation of circulating DNA from 500 2000 uL plasma serum samples The Mag Bind Circulating DNA Kit can be processed manually with 15 mL centrifuge tubes or with automated platforms using 24 well plates The procedure eliminates the needs for funnels and vacuum steps providing hands free operation in automated protocols This system combines the reversible nucleic acid binding properties of Mag Bind paramagnetic particles with a unique binding system that targets smaller DNA fragments lt 300 bp and minimizes the binding larger fragments such as gDNA If the desired target fragment is gt 300 bp please consult with your Omega Bio tek representative for a product
5. K Solution to each sample 3 Add 800 uL DCL Buffer to each sample 4 Vortex at maximum speed or pipet up and down to thoroughly mix the samples 5 Incubate at 60 C for 30 minutes Mix by inverting or shaking every 10 minutes 10 11 12 13 14 10 Mag Bind Circulating DNA Kit Protocol Add 1 8 mL ACB Buffer and 5 uL Carrier RNA Solution to each sample Vortex at maximum speed for 30 seconds or pipet up and down to mix Note ACB Buffer must be diluted with 100 isopropanol prior to use Please see Page 4 for instructions Add 100 uL Mag Bind Particles RQ Vortex at maximum speed for 10 minutes or pipet up and down to mix Continiously mix the samples throughout the 10 minute incubation period Place the plate on a magnetic separation device to magnetize the Mag Bind Particles RQ Let sit at room temperature until the Mag Bind Particles RQ are completely cleared from solution Aspirate and discard the cleared supernatant Do not disturb the Mag Bind Particles RQ Remove the plate containing the Mag Bind Particles RQ from the magnetic separation device Add 2 mL VHB Buffer to each sample Note VHB Buffer must be diluted with 100 ethanol prior to use Please see Page 4 for instructions Resuspend the Mag Bind Particles RQ by pipetting up and down 20 times or vortexing for 1 minute Note Complete resuspension of the Mag Bind Particles RQ is critical for obtaining good purity Place the pl
6. ate on the magnetic separation device to magnetize the Mag Bind Particles RQ Let sit at room temperature until the Mag Bind Particles RQ are completely cleared from solution Aspirate and discard the cleared supernatant Do not disturb the Mag Bind Particles RQ 15 16 17 18 19 20 21 22 23 24 25 26 27 Mag Bind Circulating DNA Kit Protocol Remove the plate containing the Mag Bind Particles RQ from the magnetic separation device Add 2 mL DNA Wash Buffer to each sample Note DNA Wash Buffer must be diluted with 100 ethanol prior to use Please see Page 4 for instructions Resuspend the Mag Bind Particles RQ by pipetting up and down 20 times or vortexing for 1 minute Let sit at room temperature for 1 minute Place the plate on the magnetic separation device to magnetize the Mag Bind Particles RQ Let sit at room temperature until the Mag Bind Particles RQ are completely cleared from solution Aspirate and discard the cleared supernatant Do not disturb the Mag Bind Particles RQ Repeat Steps 15 20 using 800 uL DNA Wash Buffer Remove the plate from the magnetic separation device for approximately 30 seconds Place the plate on the magnetic separation device to magnetize the Mag Bind Particles RQ Aspirate and discard the residual DNA Wash Buffer Leave the plate on thet magnetic separation device Add 500 uL water to each sample and immediately remove within 30
7. exing vigorously before use Avoid disturbing the Mag Bind Particles RQ during aspiration Increase elution volume and let sit at for 15 minutes pipet up and down 50 to 100 times Dilute DNA Wash Buffer by adding appropriate volume of ethanol prior to use see Page 4 for instructions Make sure to add ethanol to the VHB Buffer see Page 4 for instructions Dry the Mag Bind Particles RQ at 37 C for 5 minutes before elution Increase collection time on the magnet DNA Wash Buffer must be at room temperature Dry the Mag Bind Particles RQ at 37 C for 5 minutes before elution 13 Ordering Information The following components are available for purchase separately Call Toll Free at 1 800 832 8896 CAT TT Magnetic Stand for 1 5 2 0 mL tubes MSD 02 Elution Buffer EB Buffer 500 mL PD089 RNase A 400 uL AC117 RNase A 5 mL AC118 24 well Magnetic Stand A000270 14 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 Notes Notes
8. hake or vortex the Mag Bind Particles RQ to fully resuspend the particles before use The particles must be fully suspended during use to ensure proper binding Mag Bind Circulating DNA Kit Protocol Mag Bind Circulating DNA Kit Protocol for 1 2 mL Serum Plasma Materials and Reagents to be Supplied by User 100 ethanol e 100 isopropanol Magnetic separation device for 24 well plates Cat A000270 or Magnetic Separator for 15 mL centrifuge tubes Vortexer 24 well plate 10 mL Cat Whatman 7701 5102 or 15 centrifuge tubes Incubator capable of 60 C 1 5 mL microcentrifuge tubes Optional PBS or nuclease free water Before Starting Prepare ACB Buffer DNA Wash Buffer and VHB Buffer according to the Preparing Reagents section on Page 4 Set Incubator to 60 C 1 Add 1 2 mL plasma serum samples to a 10 mL 24 well plate not provided or 15 mL centrifuge tube not provided Choose the correct plasticware depending on the magnetic stand being utilized to process the samples Bring the volume up to 2 mL with Elution Buffer provided with this kit if the volume of sample is less than 2 mL 2 Add 200 uL Proteinase K Solution to each sample 3 Add 1 6 mL DCL Buffer to each sample 4 Vortex at maximum speed or pipet up and down to thoroughly mix the samples 5 Incubate at 60 C for 30 minutes Mix by inverting or shaking every 10 minutes 10 11 12 13 14 Mag Bind Circulati
9. ng DNA Kit Protocol Add 3 6 mL ACB Buffer and 10 uL Carrier RNA Solution to each sample Vortex at maximum speed for 30 seconds or pipet up and down to mix Note ACB Buffer must be diluted with 100 isopropanol prior to use Please see Page 4 for instructions Add 200 uL Mag Bind Particles RQ Vortex at maximum speed for 10 minutes or pipet up and down to mix Continiously mix the samples throughout the 10 minute incubation period Place the plate on a magnetic separation device to magnetize the Mag Bind Particles RQ Let sit at room temperature until the Mag Bind Particles RQ are completely cleared from solution Aspirate and discard the cleared supernatant Do not disturb the Mag Bind Particles RQ Remove the plate containing the Mag Bind Particles RQ from the magnetic separation device Add 3 mL VHB Buffer to each sample Note VHB Buffer must be diluted with 100 ethanol prior to use Please see Page 4 for instructions Resuspend the Mag Bind Particles RQ by pipetting up and down 20 times or vortexing for 1 minute Note Complete resuspension of the Mag Bind Particles RQ is critical for obtaining good purity Place the plate on the magnetic separation device to magnetize the Mag Bind Particles RQ Let sit at room temperature until the Mag Bind Particles RQ are completely cleared from solution Aspirate and discard the cleared supernatant Do not disturb the Mag Bind Particles RQ 15 16 17
10. seconds Remove the plate containing the Mag Bind Particles RQ from the magnetic separation device 11 28 29 30 31 32 12 Mag Bind Circulating DNA Kit Protocol Add 100 200 uL Elution Buffer or nuclease free water to elute DNA from the Mag Bind Particles RQ Resuspend the Mag Bind Particles RQ by pipetting up and down or vortexing Let sit at room temperature for 5 minutes Place the plate on the magnetic separation device to magnetize the Mag Bind Particles RQ Let sit at room temperature until the Mag Bind Particles RQ are completely cleared from solution Transfer the cleared supernatant containing purified DNA to a clean microplate or 1 5 mL microcentrifuge tube not supplied Store DNA at 20 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 800 832 8896 Problem Cause Solution Low DNA yield Mag Bind Particles RQ do not completely clear from solution Problems in downstream applications Incomplete resuspension of Mag Bind Particles RQ Loss of Mag Bind Particles RQ during operation DNA remains bound to Mag Bind Particles RQ DNA washed off Ethanol is not added into VHB buffer Ethanol carryover Too short of magnetizing time Salt carryover Ethanol carryover Resuspend the Mag Bind Particles RQ by vort
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