Mag-Bind® Stool DNA Kit - Omega Bio-Tek
Contents
1. 30 31 32 12 Mag Bind Stool DNA Kit Protocol Aspirate and discard the cleared supernatant Do not disturb the Mag Bind Particles CND Repeat Steps 20 24 for a second SPM wash step Leave the tube on the magnetic separation device for 5 10 minutes to air dry the Mag Bind Particles CND Remove any residual liquid with a pipettor Remove the tube containing the Mag Bind Particles CND from the magnetic separation device Add 50 200 uL Elution Buffer or nuclease free water Resuspend the Mag Bind Particles CND by pipetting up and down 50 times or vortexing for 3 minutes Let sit at room temperature for 5 10 minutes Note Incubation at 65 C rather than at room temperature will give a modest increase in DNA yield per elution Place the tube on the magnetic separation device to magnetize the Mag Bind Particles CND Let sit at room temperature until the Mag Bind Particles CND are completely cleared from solution Transfer the cleared supernatant containing purified DNA to a clean 1 5 mL tube Store the DNA at 20 C Note A second elution may be performed using the first eluate Mag Bind Stool DNA Kit Protocol Mag Bind Stool DNA Kit Protocol Centrifugal Protocol Note Please read through previous sections of this manual before using this protocol 1 Prepare samples by following the standard protocol in previous sections 2 Forall binding washing and elution steps centrifuge at maximum spee2. Mag Bind Stool DNA Kit Protocol Let sit on ice for 2 minutes Add 100 uL SP2 Buffer and 100 uL HTR Reagent Vortex for 30 seconds to mix thoroughly Important HTR Reagent must be thoroughly resuspended before being dispense from bottle Tip Cut the end of the pipet tips to make pipetting the HTR Reagent easier Let sit on ice for 5 minutes Centrifuge at maximum speed gt 13 000 x g for 5 minutes at room temperature Transfer 300 uL cleared supernatant to a new 1 5 mL tube Make sure not to disturb the pellet or transfer any debris Optional If RNA free DNA is required add 10 uL RNase A not supplied Incubate at 65 C for 3 minutes 10 11 12 13 14 10 Add 300 uL XP2 Buffer Vortex for 10 seconds to mix thoroughly Add 20 uL Mag Bind Particles CND and 10 uL Binding Enhancer Mix by pipetting up and down 10 20 times or vortexing for 30 seconds Let sit at room temperature for 5 minutes Mix the sample several times during incubation by vortexing or pipetting up and down 10 times Place the tube on a magnetic separation device to magnetize the Mag Bind Particles CND Let sit at room temperature until the Mag Bind Particles CND are completely cleared from solution Aspirate and discard the cleared supernatant Do not disturb the Mag Bind Particles CND 15 16 17 18 19 20 21 22 23 Mag Bind Stool DNA Kit Protocol Remove the tube containing the Mag Bind Pa
3. If using the Mag Bind Stool DNA Kit for the first time please read this booklet to become familiar with the procedures Frozen or fresh stool samples are homogenized and then lysed in a specially formulated buffer containing detergent Proteins polysaccharides and cellular debris are subsequently precipitated with SP2 Buffer after a heat freeze step Contaminants are further removed using the HTR Reagent by a quick centrifuge step Binding conditions are then adjusted and the DNA is selectively bind to the surface of Mag Bind Particles Three rapid wash steps remove trace contaminants and pure DNA is eluted in DNA Elution Buffer Purified 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 resuspension step 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 mao15 00 mao1s 01 m or5 02 Preparations 200 preps Mag Bind Particles CND 5 5 mL SLX Mlus Buffer 200 mL Storage and Stability All of the Mag Bind Stool DNA Kit components are guaranteed for at least 12 months from the date of purchase when stored as follows Mag Bind Particles CND HTR Reagent and Binding Enhancer should be stored at 2 8 C f
4. Note Complete resuspension is required for adequate washing of the Mag Bind Particles CND Place the tube on a magnetic separation device to magnetize the Mag Bind Particles CND Let sit at room temperature until the Mag Bind Particles CND are completely cleared from solution Aspirate and discard the cleared supernatant Do not disturb the Mag Bind Particles CND 26 27 28 29 30 31 32 33 Mag Bind Stool DNA Kit Protocol Repeat Steps 21 25 for a second SPM Wash Buffer wash step Leave the tube on the magnetic separation device for 5 10 minutes to air dry the Mag Bind Particles CND Remove any residual liquid with a pipettor Remove the tube containing the Mag Bind Particles CND from the magnetic separation device Add 50 200 uL Elution Buffer or nuclease free water Resuspend the Mag Bind Particles CND by pipetting up and down 50 times or vortexing for 3 minutes Let sit at room temperature for 5 10 minutes Note Incubation at 65 C rather than room temperature will give a modest increase in DNA yield per elution Place the tube on the magnetic separation device to magnetize the Mag Bind Particles CND Let sit at room temperature until the Mag Bind Particles CND are completely cleared from solution Transfer the cleared supernatant containing purified DNA to a clean 1 5 mL tube Store the DNA at 20 C Note A second elution may be performed using the first eluate Mag Bin
5. sit at room temperature until the Mag Bind Particles CND are completely cleared from solution Aspirate and discard the cleared supernatant Do not disturb the Mag Bind Particles CND Remove the tube containing the Mag Bind Particles CND from the magnetic separation device 17 18 19 20 21 22 23 24 25 Mag Bind Stool DNA Kit Protocol Add 400 uL XP2 Buffer Resuspend the Mag Bind Particles CND by vortexing or pipetting up and down 20 times Let sit at room temperature for 2 minutes Mix the sample several times during incubation by vortexing or pipetting up and down 10 times Note Complete resuspension is required for adequate washing of the Mag Bind Particles CND Place the tube on the magnetic separation device to magnetize the Mag Bind Particles CND Let sit at room temperature until the Mag Bind Particles CND are completely cleared from solution Aspirate and discard the cleared supernatant Do not disturb the Mag Bind Particles CND Remove the tube containing the Mag Bind Particles CND from the magnetic separation device Add 400 uL SPM Wash Buffer Note SPM Wash Buffer must be diluted with ethanol prior to use Please see Page 4 for instructions Resuspend the Mag Bind Particles CND by vortexing or pipetting up and down 20 times Let sit at room temperature for 1 minute Mix the sample several times during incubation by vortexing or pipetting up and down 10 times
6. Mag Bind Stool DNA Kit M4015 00 5 preps M4015 01 50 preps M4015 02 200 preps August 2012 Mag Bind Stool DNA Kit Table of Contents Introduction and OVEFVIEW csccsccsscsecssccssecssecnsecseecseccneersees 2 Kit Contents Storage and Stability 3 Preparing Rego scn ee 4 Protocol for Human DNA Detection 5 Protocol for Pathogen Detection 9 centalugal Protocol 13 Troubleshooting Guide ss 14 A eee EARR 15 Manual Revision August 2012 Fi OMEGA bio tek Innovations in nucleic acid isolation Introduction and Overview Introduction The Mag Bind Stool DNA Kit allows rapid and reliable isolation of high quality total DNA from fresh and frozen stool samples Up to 200 mg stool samples can be processed in less than 60 minutes The system combines the reversible nucleic acid binding properties of Mag Bind particles with the efficiency of HTR Reagent to eliminate humic acid polysaccharides phenolic compounds and enzyme inhibitors from stool samples Purified DNA is suitable for PCR restriction digestion and hybridization techniques There are no organic extractions thus reducing plastic waste and hands on time to allow multiple samples to be processed in parallel Overview Stool samples typically contain many compounds that can degrade DNA and inhibit downstream enzymatic reactions The Mag Bind Stool DNA Kit uses a unique HTR Reagent and SP2 Buffer that can remove inhibitory substances from stool samples
7. d 213 000 x g for 1 minute to collect the Mag Bind Particles CND instead of using the magnetic separation device 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 Problem ease Sid CT Repeat the experiment with new sample make sure the sample are completely disrupted and lysed sample stored Store the sample at 20 C incorrectly Loss of the Mag Bind Particles during operation Incomplete disruption of starting material Carefully avoid removing any Mag Bind Particles during aspiration DNA remains bound to Increase elution volume and incubate the Mag Bind Particles sample at 65 C for 10 minutes Dilute SPM Wash Buffer by adding DNA washed off appropriate volume of ethanol prior to use Page 4 rrobien cause Sotto Problems in Ethanol carry over Dry the Mag Bind Particles before elution downstream Are BSA not added to PCR Add BSA to a final concentration of 0 1 ug applications f mixture mL to the PCR mixture Low A260 280 In fficientelimination Repeat with a new sample Be sure to mix of inhibitory ratio compounds HTR Reagent thoroughly before use 14 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 tra
8. d Stool DNA Kit Protocol Mag Bind Stool DNA Kit Protocol Pathogen Detection Materials and Equipment to be Supplied by User Magnetic Separation Device for 1 5 mL Tubes Cat MSD 02 Microcentrifuge capable of at least 13 000 x g e Nuclease free 1 5 mL or 2 mL microcentrifuge tubes Water bath capable of 65 C 100 Ethanol OPTIONAL RNase A stock solution at 20 mg mL Ice bucket Before Starting Heat the water bath to 65 C Prepare the SPM Wash Buffer according to the instructions in the Preparing Reagents section on Page 4 Prepare an ice bucket 1 Weigh 50 100 mg stool sample in a 1 5 mL or 2 mL microcentrifuge tube containing 200 mg of glass beads and place the tube on ice Note If the sample is liquid pipet 200 uL sample into the microcentrifuge tube Cut the end of the pipet tip to make pipetting easier If the sample is frozen use a spatula to scrape the sample into the tube Do not thaw the frozen sample until the SLX Mlus Buffer is added into the tube 2 Add 270 uL SLX Mlus Buffer Vortex at maximum speed for 5 minutes or until the stool sample is thoroughly homogenized 3 Add 30 uL DS Buffer and 20 uL Proteinase K Solution Vortex for 10 seconds to mix thoroughly 4 Incubate at 65 C for 10 minutes 13 minutes if frozen Mix sample twice during incubation by vortexing the tube Optional For isolation of DNA from gram positive bacteria do a second incubation at 95 C for 5 minutes
9. demarks of Omega Bio tek Inc Qiagen QlAvac and Vacman are all trademarks of their respective companies PCRis a patented process of Hoffman La Roche Use of the PCR process requires a license 15 Notes
10. or long term use Proteinase K can be stored at room temperature for up to 12 months For long term storage store Proteinase K at 2 8 C During shipment or storage in cool ambient conditions precipitates may form in DS Buffer Dissolve such deposits by warming the solution at 37 C and gently shaking Preparing Reagents Dilute SPM Wash Buffer with 100 ethanol as follows and store at room temperature Porson SO CSC M4015 02 140 mL Mag Bind Stool DNA Kit Protocol Mag Bind Stool DNA Kit Protocol Human DNA Detection Materials and Equipment to be Supplied by User Magnetic Separation Device for 1 5 mL Tubes Cat MSD 02 Microcentrifuge capable of at least 13 000 x g e Nuclease free 1 5 mL or 2 mL microcentrifuge tubes Water bath capable of 65 C 100 Ethanol OPTIONAL RNase A stock solution at 20 mg mL Ice Bucket Before Starting Heat the water bath to 65 C Prepare the SPM Wash Buffer according to the instructions in the Preparing Reagents section on Page 4 Prepare an ice bucket 1 Weigh up to 200 mg stool sample in a 1 5 mL or 2 mL microcentrifuge tube and place the tube on ice 2 Add 800 uL SLX Mlus Buffer Vortex at maximum speed for 5 minutes or until the stool sample is thoroughly homogenized Note If the sample is liquid pipet 200 uL sample into the microcentrifuge tube Cut the end of the pipet tip to make pipetting easier If the sample is frozen use a spatula to scrape the
11. rticles CND from the magnetic separation device Add 400 uL XP2 Buffer Resuspend the Mag Bind Particles CND by vortexing or pipetting up and down 20 times Let sit at room temperature for 2 minutes Mix the sample several times during incubation by vortexing or pipetting up and down 10 times Note Complete resuspension is required for adequate washing of the Mag Bind Particles CND Place the tube on the magnetic separation device to magnetize the Mag Bind Particles CND Let sit at room temperature until the Mag Bind Particles CND are completely cleared from solution Aspirate and discard the cleared supernatant Do not disturb the Mag Bind Particles CND Remove the tube containing the Mag Bind Particles CND from the magnetic separation device Add 400 uL SPM Wash Buffer Note SPM Wash Buffer must be diluted with ethanol prior to use Please see Page 4 for instructions Resuspend the Mag Bind Particles CND by vortexing or pipetting up and down 20 times Let sit at room temperature for 1 minute Mix the sample several times during incubation by vortexing or pipetting up and down 10 times Note Complete resuspension is required for adequate washing of the Mag Bind Particles CND Place the tube on a magnetic separation device to magnetize the Mag Bind Particles CND Let sit at room temperature until the Mag Bind Particles CND are completely cleared from solution 11 24 25 26 27 28 29
12. sample into the tube Do not thaw the frozen sample until the SLX Mlus Buffer is added into the tube 3 Add 80 uL DS Buffer Vortex for 10 seconds to mix thoroughly 4 Centrifuge at maximum speed gt 13 000 x g for 5 minutes to pellet the stool particles 5 Transfer 600 uL supernatant into a new 1 5 mL tube Note Cut the end of the pipet tips to make pipetting easier for viscous stool samples 10 Mag Bind Stool DNA Kit Protocol Add 20 uL Proteinase K Solution Incubate at 65 C for 10 minutes Add 200 uL SP2 Buffer and 200 uL HTR Reagent Vortex for 30 seconds to mix thoroughly Important HTR Reagent must be thoroughly resuspended before being dispense from bottle Tip Cut the end of the pipet tips to make pipetting the HTR Reagent easier Let sit on ice for 5 minutes Centrifuge at maximum speed 213 000 x g for 5 minutes at room temperature Transfer 600 uL cleared supernatant to a new 1 5 mL tube Optional If RNA free DNA is required add 10 uL RNase A not supplied Incubate at 65 C for 3 minutes 11 12 13 14 15 16 Add 600 uL XP2 Buffer Vortex for 10 seconds to mix thoroughly Add 20 uL Mag Bind Particles CND Mix by pipetting 10 20 time or vortexing Let sit at room temperature for 5 minutes Mix the sample several times during incubation by vortexing or pipetting up and down 10 times Place the tube on a magnetic separation device to magnetize the Mag Bind Particles CND Let
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