Mag-Bind®Soil DNA Kit - Omega Bio-Tek
Contents
1. 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 from magnetic separation device Add 500 uL SPM Wash Buffer Vortex or pipet up and down to completely resuspend the Mag Bind Particles CND Note SPM Wash Buffer must be diluted with ethanol before use Please see Page 4 for instructions Let sit at room temperature for 2 minutes 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 Repeat Steps 27 31 for a second SPM Wash Buffer wash step Leave the tube on the magnetic separation device for 10 minutes to air dry the Mag Bind Particles CND Remove any residue liquid with a pipettor Add 50 100 uL Elution Buffer or water heated to 65 C Vortex or pipet up and down 20 times to completely resuspend the Mag Bind Particles CND Let sit at room temperature for 5 minutes 11 29 30 31 12 Mag Bind Soil DNA Kit Purification Protocol 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 Transfer2. an HiBind DNA spin column Two rapid wash steps remove trace contaminants and pure DNA is eluted in water or low ionic strength buffer Purified DNA can be directly used in downstream applications without the need for further purification New in this Edition e The latest protocol has been updated and enhanced to maximize protocol quality and readability Kit Contents Product Number Preparations 50 preps Mag Bind Particles CND 1 1mL Glass Beads 30g HTR Reagent 12 mL SLX Mlus Buffer 60 mL DS Buffer a SP2 Buffer 20 mi XP2 Buffer 30 mL VHE Buffer 22 mi Elution Buffer 20 mL SPM Wash Buffer 30 mL Storage and Stability All of the Mag Bind Soil 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 RNase A and Binding Enhancer should be stored at 2 8 C for long term use All remain ing components should be stored at room temperature During shipment or storage in cool ambient conditions precipitates may form in some buffers 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 M5635 02 140 mL per bottle Dilute VHB Buffer with 100 ethanol as follows and store at room temperature M5635 02 112 mL Mag Bind Soil DNA Kit Protocol Mag Bind Soil DNA Kit Protocol Materials and Equipment to b
3. Mag Bind Soil DNA Kit M5635 00 5 preps M5635 01 50 preps M5635 02 200 preps January 2013 Mag Bind Soil DNA Kit Table of Contents Introduction and Ove MW nica Kit Contents Storage and Stability Preparing Redge MES scsi Soil DNA Protocol use nana Soil Purification Protocol from Other Methods Troubleshooting GUI ainia a A A E E a Manual Revision January 2013 02 OMEGA bio tek Innovations in nucleic acid isolation Introduction and Overview The Mag Bind Soil DNA Kit allows rapid and reliable isolation of high quality genomic DNA from various soil samples Up to 0 25 grams of soil samples can be processed in less than 60 minutes The system combines the Mag Bind technology with HTR reagent to eliminate PCR inhibiting compounds such as humic acid from soil 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 If using the Mag Bind Soil DNA Kit for the first time please read this booklet to become familiar with the procedure Soil sample is homogenized and then treated in a specially formulated buffer Humic acid proteins polysaccharides and other contaminants are subsequently precipitated after a heat frozen step Contaminants are further removed by extraction steps Binding conditions are then adjusted and the sample is applied to
4. e Supplied by User Refrigerated microcentrifuge capable of at least 13 000 x g e 1 5 mL microcentrifuge tubes 2 ml microcentrifuge tubes Incubator capable of 70 C 100 Ethanol e 15 mL centrifuge tubes Centrifuge with rotor for 15 mL centrifuge tubes Vortexer Magnetic separation device for 1 5 mL 2 0 mL microcentrifuge tubes Before Starting Setan incubator to 70 C Heat Elution Buffer to 70 C Set an incubator or water bath to 95 C optional for gram positive bacteria Prepare ice bucket e Coola microcentrifuge to 4 C 1 Add 500 mg glass beads to a 15 mL centrifuge tube 2 Add 0 25 g soil sample 3 Add 0 6 mL SLX Mlus Buffer Vortex at maximum speed for 3 5 minutes to lyse the samples For the best result a Mixer Mill such as GenoGrinder 2010 Fastprep 24 Mixer Mill MM 3009 should be used 4 Add 60 uL DS Buffer Vortex to mix 5 Incubate at 70 C for 10 minutes Briefly vortex the tube once during incubation Optional For DNA isolation from gram positive bacteria do a second incubation at 95 C for 2 minutes 10 11 12 13 15 16 17 18 Mag Bind Soil DNA Kit Protocol Centrifuge at 13 000 x g for 3 minutes at room temperature Transfer 400 uL supernatant into a new 2 mL microcentrifuge tube Add 133 uL P2 Buffer Vortex to mix thoroughly Add 133 uL HTR Reagent Vortex to mix thoroughly Note Completely resuspend the HTR Reagent by shaking the bo
5. entrifuge tube Note If the supernatant still has a dark color from the soil repeat Steps 2 4 Add 0 5 volumes XP2 Buffer 20 uL Mag Bind Particles CND and 5 uL Binding Enhancer Pipet up and down 10 times to mix thoroughly Let sit at room temperature for 5 minutes 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 Remove the tube from magnetic separation device Add 500 uL XP2 Buffer Vortex or pipet up and down to completely resuspend the Mag Bind Particles CND Let sit at room temperature for 2 minutes 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 Remove the tube from magnetic separation device Add 500 uL VHB Buffer Vortex or pipet up and down to completely resuspend the Mag Bind Particles CND 17 18 19 20 21 22 23 24 25 26 27 28 Mag Bind Soil DNA Kit Purification Protocol Let sit at room temperature for 2 minutes Place the tube on a magnetic separation device to magnetize the Mag Bind Particles CND
6. ind Particles CND are completely cleared from solution Transfer the cleared supernatant to a new 1 5 mL microcentrifuge tube Store at 20 C Mag Bind Soil DNA Kit Purification Protocol Mag Bind Soil DNA Kit Protocol DNA Purification Protocol This protocol can be used to further purify DNA that has been isolated using other kits Materials and Equipment to be Supplied by User Refrigerated microcentrifuge capable of at least 13 000 x g 1 5 mL microcentrifuge tubes e 2mlL microcentrifuge tubes Incubator capable of 70 C 100 Ethanol 15 mL centrifuge tubes Centrifuge with rotor for 15 mL centrifuge tubes Vortexer Magnetic separation device for 1 5 mL 2 0 mL microcentrifuge tubes Optional TE Buffer pH 8 0 Before Starting Set an incubator to 70 C Heat Elution Buffer to 70 C Set an incubator or water bath to 95 C optional for gram positive bacteria Prepare ice bucket e Cool a microcentrifuge to 4 C 1 Dissolve the DNA pellet with 200 uL Elution Buffer or TE Buffer pH 8 0 2 Add 100 uL HTR Reagent Vortex to mix thoroughly Note Completely resuspend the HTR Reagent by shaking the bottle before use 3 Let sit at room temperature for 2 minutes 4 Centrifuge at maximum speed 213 000 x g for 2 minutes 10 11 12 13 14 15 16 10 Mag Bind Soil DNA Kit Purification Protocol Carefully transfer the cleared supernatant into a new microc
7. ion 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 from magnetic separation device Add 500 uL SPM Wash Buffer Vortex or pipet up and down to completely resuspend the Mag Bind Particles CND Note SPM Wash Buffer must be diluted with ethanol before use Please see Page 4 for instructions Let sit at room temperature for 2 minutes 30 31 32 33 34 35 36 37 38 Mag Bind Soil DNA Kit Protocol 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 Repeat Steps 27 31 for a second SPM Wash Buffer wash step Leave the tube on the magnetic separation device for 10 minutes to air dry the Mag Bind Particles CND Remove any residue liquid with a pipettor Add 50 100 uL Elution Buffer or water heated to 65 C Vortex or pipet up and down 20 times to completely resuspend the Mag Bind Particles CND Let sit at room temperature for 5 minutes Place the tube on a magnetic separation device to magnetize the Mag Bind Particles CND Let sit at room temperature until the Mag B
8. ore elution CTI en Littleor no supernatant Insufficient Check the centrifugal force and increase the after initial centrifugal force centrifugal time if necessary centrifuge step Salt contamination Repeat with a new sample Be sure to mix the TONAwashedoff A with SLX Mlus thoroughly Sample can not pass through Clogging column the column Check the centrifugal force and increase the time of centrifugation Ordering Information The following components are available for purchase separately Call Toll Free at 1 800 832 8896 meaa OOOO AT SPM Wash Buffer 40 mL PS014 XP2 Buffer Binding Buffer 200 mL PDR040 SP2 Buffer 60 mL PD073 14 RNase A 5 mL PD090 Notes Notes
9. the cleared supernatant to a new 1 5 mL microcentrifuge tube Store 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 1 800 832 8896 Repeat with a new sample Be sure to mix Inefficient with HTR Reagent thoroughly Add 100 uL to elimination cleared supernatant Vortex to mix Incubate of inhibitory for 2 minutes Centrifuge at 13 000 x g for 1 A seo Aoay ratio compounds minute and transfer cleared supernatant to 230 is low next step Do not reuse SP2 Buffer Repeat with a new sample Make sure the column is dried before the elution di SPW Wash Buffer wash step A A ratio is Be sure to treat the sample with RNase A za high RNA contamination p ale to the protocol Poem IN ET Low DNA Yield Poor sample or no DNA homogenization Yield DNA DNA washed off off Problem sowon BSA not added to Add BSA to a final concentration of 0 1 ug mL PCR mixture to the PCR mixture Too much DNA Dilute the DNA elute used in the downstream inhibits PCR reactions application if possible Problems in F A downstream Non specific bands in Use hot start Taq pol mixture nstre downstream PER se start Taq polymerase mixture applications Inhibitory substance Check the A A ratio in the eluted DNA Dilute the elute to 1 50 if necessary Ethanol residue in A Completely dry the column bef
10. ttle before use Let sit on ice for 5 minutes Centrifuge at 13 000 x g for for 5 minutes at 4 C Carefully transfer supernatant to a new 2 mL microcentrifuge tube Add 0 5 volumes XP2 Buffer 20 uL Mag Bind Particles CND and 5 uL Binding Enhancer Pipet up and down 10 times to mix thoroughly Let sit at room temperature for 5 minutes 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 Remove the tube from magnetic separation device Add 500 uL XP2 Buffer Vortex or pipet up and down to completely resuspend the Mag Bind Particles CND 19 20 21 22 23 24 25 26 27 28 29 Mag Bind Soil DNA Kit Protocol Let sit at room temperature for 2 minutes 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 Remove the tube from magnetic separation device Add 500 uL VHB Buffer Vortex or pipet up and down to completely resuspend the Mag Bind Particles CND Let sit at room temperature for 2 minutes Place the tube on a magnetic separat
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