E.Z.N.A.®Stool DNA Kit
Contents
1. A and MicroElute are registered trademarks of Omega Bio tek Inc Qiagen QlAvac and Vacman are all trademarks of their respective companies PCR is a patented process of Hoffman La Roche Use of the PCR process requires a license 21 Notes 22 Notes 23 Notes 242. elution Residual ethanol may interfere with downstream applications Transfer the column into a clean 1 5 mL microcentrifuge tube Add 200 uL Elution Buffer heated to 65 C directly to the center of the HiBind matrix 17 41 E Z N A Stool DNA Kit Large Volume Protocol Let sit at room temperature for 2 minutes 42 Centrifuge at maximum speed for 1 minute 43 Store DNA at 20 C 18 Note For maximum PCR robustness it is recommended to add BSA to a final concentration of 0 1 ug uL to the PCR reaction mixture Hot start PCR is also recommended to increase the specificity Try to use the minimal amount of elute possible for downstream applications 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 Inefficient elimination of Repeat with a new sample be sure to mix inhibitory compounds the sample with HTR Reagent thoroughly A__ A_ ratio Ethanol not added to ale the lysate before loading is low the column No ethanol added to Prepare DNA Wash Buffer with 100 DNA Wash Buffer ethanol A260 Maso ratiois RNA contamination Treat the sample with RNase A according high to the protocol Repeat the DNA isolation with a new sample Sample stored incorrectly Sample should be store at 4 C or 20 C Poor homogenization of Repeat with a new sample be sure to mix sample the
3. from the date of purchase when stored as follows HTR Reagent should be stored at 2 8 C for 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 All other components can 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 65 C and gently shaking Preparing Reagents Dilute DNA Wash Buffer with 100 ethanol as follows and store at room temperature D4015 02 80 mL per bottle Dilute VHB Buffer with 100 ethanol as follows and store at room temperature D4015 01 19 1 mL E Z N A Stool DNA Kit Pathogen Detection Protocol E Z N A Stool DNA Kit Protocol Pathogen Detection Materials and Equipment to be Supplied by User Microcentrifuge capable of at least 13 000 xg Nuclease free 2 mL microcentrifuge tubes Nuclease free1 5 mL microcentrifuge tubes Water baths heat blocks or incubators capable of 65 C and 70 C Vortexer 100 ethanol e Ice bath e Optional RNase A stock solution at 20 mg mL Optional Incubator capable of 95 C Before Starting Prepare ice bath Heat Elution Buffer to 65 C Set a water bath heat block or incubator to 70 C Prepare DNA Wash Buffer and VHB Buffer according to the Preparing Reagent section on Page 5 e Optional for gram positive bacteria set a
4. of 95 C Before Starting 10 Prepare ice bath Heat Elution Buffer to 65 C Set a water bath heat block or incubator to 70 C Prepare DNA Wash Buffer and VHB Buffer according to the Preparing Reagent section on Page 5 Optional for gram positive bacteria set an incubator to 95 C Add up to 200 mg stool sample in a 15 mL centrifuge tube not supplied and place the tube on ice Add 1 6 mL SLB Buffer Vortex at maximum speed for 1 minute or until the stool sample is completely homogenized Note If the sample is liquid add 200 uL sample into the centrifuge 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 SLB Buffer is added into the tube Add 180 uL DS Buffer Invert 5 times to mix Centrifuge at maximum speed gt 4 000 x g for 3 minutes Transfer 1 5 mL supernatant into a clean 15 mL centrifuge tube E Z N A Stool DNA Kit Human DNA Detection Protocol 10 11 12 13 14 13 16 17 18 19 Add 600 uL SP2 Buffer Vortex at maximum speed for 10 seconds Let sit on ice for 5 minutes Centrifuge at maximum speed 24 000 x g for 3 minutes Transfer 600 uL cleared supernatant to a new 2 mL microcentrifuge tube Add 200 uL HTR Reagent Vortex at maximum speed for 10 seconds Note HTR Reagent must be thoroughly resuspended before use Cut the end ofa 1 mL tip
5. to make it easier to pipet the HTR Reagent Let sit at room temperature for 2 minutes Centrifuge at maximum speed for 2 minutes Transfer 600 uL supernatant into a new 2 0 mL microcentrifuge tube Add 20 uL Proteinase K Solution Vortex to mix thoroughly Add 600 uL BL Buffer Vortex at maximum speed for 10 seconds Incubate at 70 C for 10 minutes Vortex the sample twice during incubation Centrifuge briefly to remove any liquid drops from the tube lid Add 600 uL 100 ethanol Vortex at maximum speed for 10 seconds Centrifuge briefly to remove any liquid drops from the tube lid Insert a HiBind DNA Mini Column into a 2 mL Collection Tube 11 E Z N A Stool DNA Kit Human DNA Detection Protocol 20 21 22 23 24 25 26 27 28 29 30 31 12 Transfer 600 uL sample from Step 18 including any precipitation that may have formed to the HiBind DNA Mini Column Centrifuge at maximum speed for 1 minute Discard the filtrate and reuse collection tube Repeat Steps 20 22 until all of the sample has been transferred to the HiBind DNA Mini Column Transfer the HiBind DNA Mini Column to a new 2 mL Collection Tube Add 500 uL VHB Buffer Note VHB Buffer must be diluted with ethanol before use Please see the Preparing Reagents section on Page 5 for instructions Centrifuge at maximum speed for 30 seconds Discard the filtrate and reuse collection tube Add 700
6. E Z N A Stool DNA Kit D4015 00 5 preps D4015 01 50 preps D4015 02 200 preps April 2013 E Z N A Stool DNA Kit Table of Contents Introduction and OyervieWi css 2 Illustrated Protocol une 3 Kit Contents Storage and Stability 4 Preparing Regencia 5 Stool DNA Protocol Pathogen Detection 6 Stool DNA Protocol Human DNA Detection 10 Stool DNA Protocol Large Volume 14 Troubleshooting CU dera 19 OTERO nara 21 Manual Revision April 2013 Fi OMEGA bio tek Innovations in nucleic acid isolation Introduction and Overview The E Z N A 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 our HiBind matrix with the speed and versatility of spin column technology 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 allowing multiple samples to be processed in parallel Stool samples typically contain many compounds that can degrade DNA and inhibit downstream enzymatic reactions E Z N A Stool DNA Kit uses an unique HTR Reagent and P2 Buffer that can remove inhibitory substances from stool samples If
7. L DS Buffer Incubate at 70 C for 10 minutes Centrifuge at maximum speed gt 4 000 x g for 15 minutes Transfer the supernatant to a new 15 mL or 50 mL centrifuge tube Note To make pipetting easier for viscous stool samples cut the end of the pipet tips Add 1 3 volume SP2 Buffer Vortex at maximum speed for 10 seconds Let sit on ice for 5 minutes Centrifuge at maximum speed gt 13 000 x g for 10 minutes Transfer the cleared supernatant to a new 15 or 50 mL centrifuge tube Add 1 volume isopropanol Invert the tube 10 times to mix Centrifuge at maximum speed for 10 minutes Discard the supernatant and invert the tube on a absorbent paper to drain the liquid drops Add 250 uL Elution Buffer Vortex at maximum speed for 20 seconds Incubate at 70 C for 10 20 minutes Vortex the sample twice during incubation 15 E Z N A Stool DNA Kit Large Volume Protocol Optional If RNA free DNA is required add 10 uL RNase A Vortex to mix thoroughly 16 Add 200 uL HTR Reagent Vortex at maximum speed for 10 seconds Note HTR Reagent must be thoroughly resuspended before use Cut the end of a 1 mL tip to make it easier to pipet the HTR Reagent 17 Let sit at room temperature for 2 minutes 18 Centrifuge at maximum speed for 2 minutes 19 Transfer 250 uL supernatant into a new 1 5 mL microcentrifuge tube 20 Add 10 uL Proteinase K Vortex to mix thoroughly 21 Add 250 uL BL Buffer Vortex at maximum speed for 10 se
8. card the filtrate and reuse collection tube Repeat Steps 22 24 for a second DNA Wash Buffer wash step E Z N A Stool DNA Kit Pathogen Detection Protocol 26 27 28 29 30 31 Centrifuge at maximum speed for 2 minutes to dry the column Note It is important to dry the column membrane before elution Residual ethanol may interfere with downstream applications Transfer the column into a clean 1 5 mL microcentrifuge tube Add 100 200 uL Elution Buffer heated to 65 C directly to the center of the HiBind matrix Let sit at room temperature for 2 minutes Centrifuge at maximum speed for 1 minute Store DNA at 20 C Note For maximum PCR robustness it is recommended to add BSA to a final concentration of 0 1 ug uL to the PCR reaction mixture Hot start PCR is also recommended to increase the specificity Try to use the minimal amount of elute possible for downstream applications E Z N A Stool DNA Kit Human DNA Detection Protocol E Z N A Stool DNA Kit Protocol Human DNA Detection Materials and Equipment to be Supplied by User Centrifuge with adaptor for 15 mL centrifuge tubes capable of 4 000 x g Microcentrifuge capable of at least 13 000 xg Nuclease free 1 5 mL and 2 mL microcentrifuge tubes 15 mL centrifuge tubes Water baths heat blocks or incubators capable of 65 C and 70 C Vortexer 100 ethanol Ice bath Optional RNase A stock solution at 20 mg mL Optional Incubator capable
9. conds 22 Incubate at 70 C for 5 minutes Vortex the sample twice during incubation 23 Centrifuge briefly to remove any liquid drops from the tube lid 24 Add 250 uL 100 ethanol Vortex at maximum speed for 10 seconds 25 Centrifuge briefly to remove any liquid drops from the tube lid 26 Inserta HiBind DNA Mini Column into a 2 mL Collection Tube 27 Transfer the entire sample from Step 13 including any precipitates that may have formed to the HiBind DNA Mini Column 28 Centrifuge at maximum speed for 1 minute 16 29 30 31 32 33 34 35 36 37 38 39 40 E Z N A Stool DNA Kit Large Volume Protocol Discard the filtrate and collection tube Transfer the HiBind DNA Mini Column to a new 2 mL Collection Tube Add 500 uL VHB Buffer Note VHB Buffer must be diluted with ethanol before use Please see the Preparing Reagents section on Page 5 for instructions Centrifuge at maximum speed for 30 seconds Discard the filtrate and reuse collection tube Add 700 uL DNA Wash Buffer Note DNA Wash Buffer must be diluted with ethanol before use Please see the Preparing Reagents section on Page 5 for instructions Centrifuge at maximum speed for 1 minute Discard the filtrate and reuse collection tube Repeat Steps 28 30 for a second DNA Wash Buffer wash step Centrifuge at maximum speed for 2 minutes to dry the column Note It is important to dry the column membrane before
10. esuspended before use Cut the end of a 1 mL tip to make it easier to pipet the HTR Reagent 10 Let sit at room temperature for 2 minutes 11 Centrifuge at maximum speed for 2 minutes 12 Transfer 250 uL supernatant to a new 1 5 mL microcentrifuge tube Optional If RNA free DNA is required add 10 uL RNase A not included Vortex to mix thoroughly Incubate at 37 C for 3 minutes Continue to Step 13 13 Add 250 uL BL Buffer and 250 uL 100 ethanol Vortex at maximum speed for 10 seconds E Z N A Stool DNA Kit Pathogen Detection Protocol 14 15 16 17 18 19 20 21 22 23 24 25 Insert a HiBind DNA Mini Column into a 2 mL Collection Tube Transfer the entire sample from Step 13 including any precipitates that may have formed to the HiBind DNA Mini Column Centrifuge at maximum speed for 1 minute Discard the filtrate and the collection tube Transfer the HiBind DNA Mini Column into a new 2 mL Collection Tube Add 500 uL VHB Buffer Note VHB Buffer must be diluted with ethanol before use Please see the Preparing Reagents section on Page 5 for instructions Centrifuge at maximum speed for 30 seconds Discard the filtrate and reuse the collection tube Add 700 uL DNA Wash Buffer Note DNA Wash Buffer must be diluted with ethanol before use Please see the Preparing Reagents section on Page 5 for instructions Centrifuge at maximum speed for 1 minute Dis
11. n incubator to 95 C 1 Add up to 200 mg stool sample in a 2 mL microcentrifuge tube containing 200 mg glass beads Place the tube on ice Note If the sample is liquid add 200 uL sample into the centrifuge 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 SLB Buffer is added into the tube 2 Add 540 uL SLB Buffer Vortex at maximum speed for 10 minutes or until the stool sample is thoroughly homogenized Note We recommend a mechanical disruptor instrument such as the SPEX Geno Grinder 2010 or a flat bed vortexer with tape 3 Add 60 uL DS Buffer and 20 uL Proteinase K Solution Vortex or pipet up and down to mix thoroughly E Z N A Stool DNA Kit Pathogen Detection Protocol 4 Incubate at 70 C for 10 minutes 13 minutes if frozen Vortex the sample twice during incubation Optional For isolation of DNA from gram positive bacteria do a second incubation at 95 C for 5 minutes Continue to Step 5 5 Add 200 uL SP2 Buffer Vortex at maximum speed for 30 seconds 6 Let sit on ice for 5 minutes 7 Centrifuge at full speed 213 000 x g for 5 minutes 8 Carefully aspirate 400 uL supernatant to a new 1 5 mL microcentrifuge tube not supplied Do not to disturb the pellet or transfer any debris 9 Add 200 uL HTR Reagent Vortex at maximum speed for 10 seconds Note HTR Reagent must be thoroughly r
12. rces in the stool sample Please note that excess volume of reagents will be required to use this protocol Additional reagents can be purchased separately call Omega Bio tek at 1 800 832 8896 for more information Materials and Equipment to be Supplied by User Centrifuge with adaptor for 15 mL or 50 mL centrifuge tubes capable of 4 000xg Microcentrifuge capable of at least 13 000xg Nuclease free 1 5 mL and 2 mL microcentrifuge tubes 15 mL or 50 mL centrifuge tubes Water baths heat blocks or incubators capable of 65 C and 70 C Vortexer 100 ethanol Isopropanol Ice bath Optional RNase A stock solution at 20 mg mL Optional Incubator capable of 95 C Before Starting 14 Prepare ice bath Heat Elution Buffer to 65 C Set a water bath heat block or incubator to 70 C Prepare DNA Wash Buffer and VHB Buffer according to the Preparing Reagent section on Page 5 Optional for gram positive bacteria set an incubator to 95 C Add up to 2 g stool sample to a 15 mL or 50 mL centrifuge tube and place the tube on ice Add 10 volumes SLB Buffer Vortex at maximum speed for 1 minute or until the stool sample is thoroughly homogenized Note For example add 10 mL SLB Buffer to 1 g stool sample 10 11 12 13 14 15 E Z N A Stool DNA Kit Large Volume Protocol Add 1 10 volume DS Buffer and 20 uL Proteinase K Solution Mix by inverting 10 times Note For example if 10 mL SLB Buffer was used add 1 m
13. sample with SLB Buffer thoroughly Low DNA yield or no DNA DNA washed off eluted Dilute DNA Wash Buffer with 100 ethanol prior to use Page 5 Add 100 uL 3M NaOH to the column prior to loading the sample Centrifuge at 10 000 x g for 30 seconds Add 100 uL water to the columns and centrifuge at 10 000 x g for 30 seconds Discard the filtrate Column matrix loses binding capacity during storage 19 Troubleshooting Guide BSA not added to PCR Add BSA to a final concentration of 0 1 mixture ug mL to the PCR mixture Too much DNA inhibits Dilute the eluted DNA before use if Problems in PCR reactions possible downstream N ande FR on specific bands in applications Use hot start Taq polymerase mixture downstream PCR q poy Inhibitory substance in Check the A A ratio 260 230 the eluted DNA Dilute the elute 1 50 if necessary Ethanol residue in elute Completely dry column before elution Little or no supernatant Insufficient centrifugal Check the centrifugal force and increase after initial force the centrifugal time if necessary centrifuge step Sample can not passthrough Clogged column the column Check the centrifugal force and increase the time of centrifugation 20 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 SP2 Buffer 60 mL PD073 HiBind E Z N
14. uL DNA Wash Buffer Note DNA Wash Buffer must be diluted with ethanol before use Please see the Preparing Reagents section on Page 5 for instructions Centrifuge at maximum speed for 30 seconds Discard the filtrate and reuse collection tube Repeat Steps 28 30 for a second DNA Wash Buffer wash step E Z N A Stool DNA Kit Human DNA Detection Protocol 32 33 34 35 36 37 Centrifuge at maximum speed for 2 minutes to dry the column Note It is important to dry the column membrane before elution Residual ethanol may interfere with downstream applications Transfer the column into a clean 1 5 mL microcentrifuge tube Add 100 200 uL Elution Buffer heated to 65 C directly to the center of the HiBind matrix Let sit at room temperature for 2 minutes Centrifuge at maximum speed for 1 minute Store DNA at 20 C Note For maximum PCR robustness it is recommended to add BSA to a final concentration of 0 1 ug uL to the PCR reaction mixture Hot start PCR is also recommended to increase the specificity Try to use the minimal amount of elute possible for downstream applications 13 E Z N A Stool DNA Kit Large Volume Protocol E Z N A Stool DNA Protocol for Large Volumes of Stool The following protocol is designed when the targeting DNA is not distributed homogeneously in the stool sample Using large volumes of starting material will enhance the chances of isolating DNA from lower titer sou
15. using the E Z N A 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 P2 Buffer after a heat freeze step Contaminants are further removed by HTR Reagent during a quick centrifuge step Binding conditions are adjusted by adding BL Buffer and the sample is applied to a HiBind DNA Mini Column Two rapid wash steps remove trace contaminants and pure DNA is eluted with Elution Buffer Purified DNA can be directly used in downstream applications without the need for further purification New in this Edition e 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 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 O ab 0 EN ao Illustrated Protocol Lyse Samples Precipitate Polysaccharides Remove Inhibitors Transfer Supernatant and Bind DNA Wash 2X Dry Elute Kit Contents Storage and Stability All of the E Z N A Stool DNA Kit components are guaranteed for at least 12 months
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