E.Z.N.A.®Soil DNA Kit
Contents
1. Poor homogenization of sample be sure to vortex the sample sample with SLX Mlus and glass beads thoroughly DNA washed off SPW Wash Buffer must be diluted with ethanol before use Add 100 uL 3M NaOH to the column prior to loading the sample Centrifuge at 10 000 x g for 30 seconds Discard the filtrate Salt contamination Column matrix lost binding capacity during storage BSA not added to PCR Add BSA to a final concentration of mixture 0 1 ug mL to the PCR mixture Too much DNA inhibits Dilute the DNA used in the down PCR reactions stream application if possible Non specific bands in Use hot start Taq polymerase mix downstream PCR ture Inhibitory substance in Check the A ration the eluted DNA Dilute the elute to 1 50 if necessary Residual ethanol in the Completely dry the column before elute elution Check the centrifugal force and increase the centrifugal time if necessary Insufficient centrifugal force Check the centrifugal force and Clogged column i a F 99 increase the time of centrifugation Ordering Information The following components are available for purchase separately Call Toll Free at 1 800 832 8896 HiBind DNA Mini Columns 200 columns DNACOL 02 P2 Buffer 60 mL PD076 SPW Wash Buffer 40 mL PDRO45 Elution Buffer 100 mL PDRO48 HiBind E Z N A and MicroElute are registered trademarks of Omega Bio tek Inc PCR is a patented process of Hoffman La Roche U2. transfer the supernatant to anew 2 mL microcentrifuge tube Add 0 7 volumes isopropanol Mix thoroughly by inverting tube for 20 30 times Note If the soil contains very low DNA incubate the sample at 20 C for 1 hour Centrifuge at gt 13 000 x g for 10 minutes at 4 C Carefully aspirate and discard the supernatant Do not disturb the DNA pellet Invert the tube on a absorbent paper for 1 minute to drain the liquid Note It is not necessary to dry the DNA pellet Add 200 uL Elution Buffer Vortex for 10 seconds Incubate at 70 C for 10 20 minutes to dissolve the DNA pellet 18 19 20 21 22 23 24 25 26 27 28 29 30 E Z N A Soil DNA Kit Protocol Add 100 uL HTR Reagent Vortex to mix thoroughly Note Completely resuspend HTR Reagent by shaking the bottle before use Let sit at room temperature for 2 minutes Centrifuge at gt 13 000 x g for 2 minutes Transfer cleared supernatant to a new 2 mL microcentrifuge tube Note If supernatant still has a dark color from the soil repeat Steps 18 20 for a second HTR Reagent step Add an equal volume XP1 Buffer Vortex to mix thoroughly Insert a HiBind DNA Mini Column into a 2 mL Collection Tube provided in this kit Transfer the sample from Step 22 to the HiBind DNA Mini Column Centrifuge at 10 000 x g for 1 minute at room temperature Discard the filtrate and reuse the Collection Tube Add 300 uL XP1 Buff
3. E Z N A Soil DNA Kit D5625 00 5 preps D5625 01 50 preps D5625 02 200 preps April 2013 E Z N A Soil DNA Kit Table of Contents Introduction and DyeiievW nn 2 Kit Contents Storage and Stabil une 3 Preparing Reagan ee 4 SOlLDNA Prot COL 5 Purification of DNA Isolated using Other Methods 9 Troubleshooting Gulden 10 ONE NG ee were 11 Manual Revision April 2013 Fi OMEGA bio tek Innovations in nucleic acid isolation Introduction and Overview Introduction The E Z N A Soil DNA Kit allows rapid and reliable isolation of high quality genomic DNA from various soil samples Up to 1 gram of soil samples can be processed in less than 60 minutes The system combines the reversible nucleic acid binding properties of HiBind matrix with the speed and versatility of spin column technology 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 Overview If using the E Z N A 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 containing detergent Humic acid proteins polysaccharides and other contaminants are subsequently precipitated after a heat fr
4. eeze step Contaminants are further removed by extraction steps Binding conditions are then adjusted and the sample is applied to an HiBind DNA Mini 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 Equilibration Buffer used in the Troubleshooting section is no longer included with this kit Equilibration Buffer can be replaced with 3M NaOH provided by the user Kit Contents Product Number D5625 02 Purifications 200 preps HiBind DNA Mini Columns 2 mL Collection Tubes 400 SPW Wash Buffer Storage and Stability Most components of the E Z N A Soil DNA Kit should be stored at 22 25 C Store the HTR Reagent at 2 8 C Under these conditions DNA has successfully been purified and used for PCR after 24 months of storage During shipment or storage in cool ambient conditions precipitates may form in some buffers It is possible to dissolve such deposits by warming the buffer to 55 C Preparing Reagents Dilute SPW Wash Buffer with 100 ethanol as follows and store at room temperature D5625 02 100 mL per bottle E Z N A Soil DNA Kit Protocol E Z N A Soil DNA Protocol Materials and Equipment to be Supplied by User Microcentrifuge capable of at least 13 000 x g and 4 C e Centrifuge with rotor for 15 mL ce
5. er Centrifuge at 10 000 x g for 1 minute Discard the filtrate and the Collection Tube Transfer the HiBind DNA Mini Column into a new 2 mL Collection Tube 31 32 33 34 35 36 37 38 39 40 41 E Z N A Soil DNA Kit Protocol Add 700 uL SPW Wash Buffer Note SPW Wash Buffer must be diluted with ethanol before use Please see the Preparing Reagents section on Page 4 for instructions Centrifuge at 10 000 x g for 1 minute Discard the filtrate and reuse the Collection Tube Repeat Steps 31 33 for a second SPW Wash Buffer wash step Centrifuge the empty HiBind DNA Mini Column at gt 13 000 x g for 2 minutes at room temperature Note This step is critical in removing residual ethanol that may interfere with downstream applications Transfer the HiBind DNA Mini Column into a clean 1 5 mL microcentrifuge tube Add 30 100 uL Elution Buffer heated to 70 C directly onto the center of HiBind membrane Incubate at 70 C for 10 15 minutes Centrifuge at gt 13 000 x g for 1 minute Repeat Steps 37 39 for a second elution step Discard the HiBind DNA Mini Column and store eluted DNA at 20 C E Z N A Soil DNA Kit Protocol E Z N A Soil DNA Protocol Purification of DNA Isolated using Other Methods Materials and Equipment to be Supplied by User Microcentrifuge capable of at least 13 000 x g and 4 C 1 5 mL microcentrifuge tubes 2 ml microcentrifuge tub
6. es Incubator capable of 70 C 100 ethanol Before Starting Prepare the SPW Wash Buffer as instructed in the Preparing Reagents section on Page 4 Heat Elution Buffer to 70 C Seta incubator to 70 C Seta microcentrifuge to 4 C 1 Adjust the volume of the DNA sample to 200 uL with Elution Buffer 2 Add 100 uL HTR Reagent Vortex to mix thoroughly Note Completely resuspend HTR Reagent by shaking the bottle before use 3 Let sit at room temperature for 2 minutes 4 Centrifuge at gt 13 000 x g for 2 minutes 5 Transfer cleared supernatant to a new 2 mL microcentrifuge tube Note If supernatant still has a dark color from the soil repeat Steps 2 4 for a second HTR Reagent step 6 Follow Steps 22 41 beginning on Page 7 of the Soil DNA Protocol 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 A260 230 ratio is low Low DNA Yield or no DNA Yield Problems in downstream applications Little or no supernatant after initial centrifuge step Sample can not pass through the column Repeat with a new sample be sure to mix the sample with HTR Reagent thoroughly Inefficient elimination of inhibitory compounds Make sure the column is dried before the elution Wash the column with extra SPW Wash Buffer Repeat the DNA isolation with a new
7. ntrifuge tubes Vortexer 1 5 mL microcentrifuge tubes 2 ml microcentrifuge tubes e 15 mL centrifuge tubes Incubator capable of 70 C 100 ethanol lsopropanol Optional Incubator capable of 95 C Before Starting Prepare the SPW Wash Buffer as instructed in the Preparing Reagents section on Page 4 Heat Elution Buffer to 70 C Seta incubator to 70 C Prepare ice bucket Seta microcentrifuge to 4 C e Optional For gram positive bacteria set a incubator or water bath to 95 C 1 Transfer 500 mg glass beads to a 15 mL centrifuge tube 2 Add 0 2 1 0 g soil sample to the glass beads 3 Add 1 mL SLX Mlus Buffer Vortex at maximum speed for 3 5 minutes to lyse samples Note For best result a mixer mill such as GenoGrinder 2010 Fastprep 24 Mixer Mill MM 300 should be used 4 Add 100 uL DS Buffer Vortex to mix thoroughly 5 Incubate at 70 C for 10 min Briefly vortex the tube once during the incubation E Z N A Soil DNA Kit Protocol Optional For isolation of DNA from gram positive bacteria do a second incubation at 95 C for 2 minutes 6 10 11 12 13 14 15 16 17 Centrifuge at 3 000 rpm for 3 minutes at room temperature Transfer 800 uL the supernatant into anew 2 mL microcentrifuge tube Add 270 uL P2 Buffer Vortex to mix thoroughly Incubate on ice for 5 minutes Centrifuge at gt 13 000 x g for 5 minutes at 4 C Carefully
8. se of the PCR process requires a license 11 Notes
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