PSP Spin Stool DNA Kit/ PSP Spin Stool DNA Plus Kit User manual
Contents
1. Equipment and reagents to be supplied by user When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information please consult the appropriate material safety data sheets MSDS See our webpage www stratec com Microcentrifuge Thermomixer for 95 C Measuring cylinder 250 ml Disposable gloves Pipet with tips Reagents reservoirs for multichannel pipets 96 100 ethanol ddH2O0 Vortexer or other homogenizer Isopropanol O O OO oO OO 0 O 6 The PSP Spin Stool DNA Kit and the PSP Spin Stool DNA Plus Kit are validated with 2 Propanol Rotipuran gt 99 7 p a ACS ISO Order no 6752 from Carl Roth Possible suppliers for lsopropanol Carl Roth 2 Propanol Applichem Sigma Rotipuran gt 99 7 p a ACS ISO 2 Propanol f r die Molekularbiologie 2 Propanol Order no 6752 Order no A3928 Order no 59304 1L F 13 PSP Spin Stool DNA Kit 0515 PSP Spin Stool DNA Plus Kit 0515 Important indications 1 The kit procedure is also suitable for purifying DNA from very small amounts of starting material If the sample has less than 5 ng DNA gt 1 000 copies 3 5 ug Carrier a homopolymer such as poly dA poly dT or genomic DNA should be added to the starting material Ensure that the Carrier DNA does not interfere with downstream application In order to prevent any interference of the carrier with the downstream application a RNA carrier c2. stratecee molecular User manual a PSP Spin Stool DNA Kit PSP Spin Stool DNA Plus Kit for collection storage stabilization and purification of total DNA from fresh or frozen stool samples including Stool Collection Tubes with Stool DNA Stabilizer c 1038100x00 1038110x00 geal STRATEC Molecular GmbH D 13125 Berlin Instruction for the PSP Spin Stool DNA Kit The PSP Spin Stool DNA Kit provides fast and easy purification of total DNA from max 200 mg of fresh or frozen stool samples using the Invisorb technology The purified DNA is of high quality and well suited for use in in vitro diagnostic analysis Instruction for PSP Spin Stool DNA Plus Kit The PSP Spin Stool DNA Plus Kit is an integrated system for collection transportation and storage of stool samples and subsequent DNA purification The kit has been designed for isolation of DNA from pathogenic microorganisms as well as for isolation of DNA from the host organism Furthermore it is possible to extract nucleic acids from food and feed residues of plant or animal origin from the stool sample The purified DNA is ideal for reliable use in PCR and other downstream enzymatic reactions The kits are neither validated for the isolation of genomic DNA from cultured or isolated cells from tissues swabs dried blood stains or cell free body fluids like synovial fluid and urine or the purification of RNA The application of the kits for
3. Open the Stool Collection Tube and collect a spoon of the stool sample 2 Transfer the spoon with the stool sample back into the Stool Collection Tube and close the tube very tight 3 Mix the sample for a short time by shaking That will lead to homogenization of the stool sample Important Notes The collected sample can be stored at ambient temperature for at least 3 days The storage under Stool DNA Siabilizer will lead to a better yield of bacterial pathogens with difficult to lyse cell walls Storage time has no influence on the quality or the amount of host cell DNA The collected sample can also be used immediately after collection for the isolation of DNA The collected sample can be frozen at 20 C immediately after collection or after storage at ambient temperature for a later use for example for a second DNA isolation 21 PSP Spin Stool DNA Kit 0515 PSP Spin Stool DNA Plus Kit 0515 Protocol 2 Isolation of total DNA from 1 4 ml stabilized stool homogenate with and without enrichment of bacterial DNA Please read protocols prior the start of the preparation and complete preparing steps Attention Please be aware that you have to prepare the Binding Buffer A see instruction page 13 Important Note Please note that the extracted DNA from stool sample is by the majority from bacterial origin Heat heating blocks e g thermomixer to 70 C and 95 Preheat the Elution Buffer to 70 e g transfer the ne
4. mix for a short time by shaking transfer 1 4 ml of the stabilized stool sample Stool DNA Stabilizer with stool specimen into a 2 0 ml Safe Lock Tube for enrichment of host DNA incubate 10 min at RT under shaking for enrichment of bacterial DNA incubate 10 min at 95 C on a thermomixer under shaking add 5 Zirconia Beads II to the homogenate and vortex for 2 min spin down at 11 000 x g 11 000 rpm for 1 min transfer the supernatant to the InviAdsorb Tube mix it by vortexing for 15 sec incubate 1 min at RT spin down for 3 min at full speed transfer the supernatant in a new 1 5 ml Receiver Tube centrifuge the sample again at full speed for 3 min add 25 ul Proteinase K in a new 2 0 ml Safe Lock Tube transfer 800 ul of the sample supernatant to the same tube mix shortly by vortexing incubate for 10 min at 70 C while continuously shaking on a thermomixer at 900 rpm add 400 ul Binding Buffer A follow preparing instructions to the lysate mix shortly by vortexing or pipetting up and down transfer the whole mixture in two steps to the RTA Spin Filter incubate for 1 min at RT centrifuge at 11 000 x g 11 000 rpm for 2 min discard the filtrate and the RTA Receiver Tube transfer the RTA Spin Filter in a new RTA Receiver Tube pipet 500 ul Wash Buffer I onto the RTA Spin Filter centrifuge at 11 000 x g 11 000 rpm for 1 min discard the flow through and the RTA Receiver Tube put the RTA Spin Filter in a new RTA Receiv
5. 95 C thermo block Incubate for further 3 min at 95 C Vortex the sample for 2 min Centrifuge the sample at 11 000 x g 11 000 rpm for 1 min to pellet solid stool particles 2 Removal of PCR Inhibitiors Transfer the supernatant into an InviAdsorb Tube and vortex vigorously for 15 sec Incubate the suspension for 1 min at room temperature Centrifuge the sample at full speed for 3 min 3 Second Sample Cleanup Transfer the supernatant completely into a new 1 5 ml Receiver Tube Discard the pellet Centrifuge the sample again at full soeed for 3 min 4 Proteinase K digestion Transfer 25 ul Proteinase K into a new 2 0 ml Safe Lock Tube and pipet 400 ul of the supernatant from step 3 to the 1 5 ml Receiver Tube containing Proteinase K mix shortly by vortexing and incubate the sample for 10 min at 70 C in a thermomixer under continuous shaking at 900 rpm 18 PSP Spin Stool DNA Kit 0515 PSP Spin Stool DNA Plus Kit 0515 5 Binding of the DNA Add 200 ul of Binding Buffer A to the lysate and mix shortly by vortexing or by pipetting up and down several times Transfer the mixture completely onto the membrane of the RTA Spin Filter Incubate for 1 min at room temperature and centrifuge at 11 000 x g 11 000 rpm for 2 min Discard the filtrate and the RTA Receiver Tube 6 Washing Steps Put the RTA Spin Filter in a new RTA Receiver Tube Add 500 ul Wash Buffer I to the membrane of the RTA Spin Filter Close the Close the l
6. Kit 0515 Procedure Lysis Stool samples are lysed in Lysis Buffer P under denaturing conditions at high temperatures Human cells lyse efficiently at RT bacterial cells and those of other pathogens in the stool sample are efficiently lysed by incubation at 95 C This is recommended for detection of cells that are difficult to lyse e g gram positive bacteria Note The total DNA concentration in the lysate will be increased 3 5 fold by lysis at 95 C and the ratio of non human to human DNA will increase Removal of PCR inhibitors After lysis procedure DNA damaging substances and PCR inhibitors which are present in the feces are adsorbed efficiently to the InviAdsorb matrix InviAdsorb is provided very convenient in safe lock tubes and the lysate must only be mixed with the matrix The bound contaminations and cell debris are pelleted by centrifugation The supernatant contains the precleaned DNA Protein digestion Proteinase K is added to the supernatant to digest and degrade proteins during the incubation at 70 C Binding of total DNA After adding Binding Buffer A to the supernatant the mixture is transferred to the spin columns and nucleic acids are bound to the membrane of the RTA Spin Filter during a brief centrifugation step Optimal salt concentrations and pH conditions in the lysate ensure that remains of digested proteins and other contaminations which can inhibit downstream enzymatic reactions are not retained on the I
7. add 100 200 ul preheated 70 C Elution Buffer Incubate for 1 min Centrifuge at 11 000 x g 11 000 rpm for 1 min to elute the DNA Finally discard the RTA Spin Filter Note The DNA can also be eluted with a lower volume of Elution Buffer depends on the expected yield of genomic DNA But pay attention that the minimum volume for the elution is 50 ul and that this volume can reduce the maximum yield If a quite large amount of DNA is expected the volume of elution can be increased Note For long term storage we recommend to keep the eluted DNA at 20 TC 23 PSP Spin Stool DNA Kit 0515 PSP Spin Stool DNA Plus Kit 0515 Protocol 3 solation of total DNA from up to 400 mg stool samples from difficult to lyse bacteria Please read protocols prior the start of the preparation and complete preparing steps Attention Please be aware that you have to prepare the Binding Buffer A see instruction page 13 Important Note To lyse some special bacteria completely like Mycobacteria paratuberculosis or Chlamydia a special treatment is needed Heat heating blocks e g thermomixer to 70 C and 95 Prepare a container with crashed ice Preheat the Elution Buffer to 70 e g transfer the needed volume into a tube and place it at the appropriate temperature into a thermomixer 1 Sample Homogenization and Prelysis Transfer 1 4 ml of the collected and well homogenized stool sample Stool DNA Stabilizer with stool specimen
8. always keep the bottle firmly closed Add 105 ml of 96 100 ethanol to each bottle Wash Buffer Il mix thoroughly and always keep the bottle firmly closed Preheat needed amount of Elution Buffer at 70 C ina thermomixer PSP Spin Stool DNA Kit 0515 PSP Spin Stool DNA Plus Kit 0515 Symbols Manufacturer Lot number Catalogue number Expiry date Consult operating instructions Temperature limitation e Bla Be Do not reuse Storage All buffers and kit contents of the PSP Spin Stool DNA Kit and the PSP Spin Stool DNA Plus Kit except dissolved Proteinase K should be stored at room temperature RT and are stable for at least 12 months under these conditions Room temperature RT is defined as range from 15 30 C Proteinase K Dissolved Proteinase K must be stored at 20 C Repeated freezing and thawing will reduce the activity of Proteinase K dramatically Dividing Proteinase K into aliquots and store at 20 C Wash Buffer and Il Wash Buffer charged with ethanol should be stored at room temperature and should be sealed appropriately If there are any precipitates within the provided solutions redissolve these precipitates by careful warming up to room temperature up to 30 C Quality control and product warranty STRATEC Molecular warrants the correct function of the PSP Spin Stool DNA Kit and the PSP Spin Stool DNA Plus Kit for applications as described in this manual Purchaser must deter
9. are necessary O O O to avoid cross contamination between sample preparation when handling RTA Spin Filter Set carefully apply the sample or solution to the RTA Spin Filter Set pipet the sample into the filter without wetting the rim of the column always change pipet tips between liquid transfers we recommend the use of aerosol barrier pipet tips avoid touching the RTA Spin Filter membrane with the pipet tip 14 PSP Spin Stool DNA Kit 0515 PSP Spin Stool DNA Plus Kit 0515 Scheme of the PSP Spin Stool DNA Kit Please read protocols prior the start of the preparation carefully transfer 200 mg of the stool sample into a 2 ml Safe Lock Tube add 1 2 ml Lysis Buffer P vortex for 1 min for enrichment of host DNA incubate 10 min at RT under shaking for enrichment of bacterial DNA incubate 10 min at 95 C on a thermomixer under shaking add 5 Zirconia Beads II to the homogenate and vortex for 2 min spin down at 11 100 x g 11 000 rpm for 1 min transfer the supernatant to the InviAdsorb Tube mix it by vortexing for 15 sec incubate 1 min at RT spin down for 3 min at full speed transfer the supernatant in a new 1 5 ml Receiver Tube centrifuge the sample again at full soeed for 3 min transfer 400 ul of the sample supernatant in a new 1 5 ml Receiver Tube add 25 ul Proteinase K to the same 2 0 ml Safe Lock Tube mix shortly by vortexing incubate for 10 min at 70 C while continuously shaking on a thermo
10. determined from the concentration of DNA in the eluate measured by absorbance at 260 nm Purity is determined by calculating the ratio of absorbance at 260 nm to absorbance at 280 nm Pure DNA has an A260 A280 ratio of 1 7 2 1 Absorbance readings at 260 nm should lie between 0 1 and 1 0 to be accurate Sample dilution should be adjusted accordingly Use elution buffer or water as appropriate to dilute samples and to calibrate the spectrophotometer Measure the absorbance at 260 and 280 nm or scan absorbance from 220 320 nm a scan will show if there are other factors affecting absorbance at 260 nm Both DNA and RNA are measured with a spectrophotometer 30 PSP Spin Stool DNA Kit 0515 PSP Spin Stool DNA Plus Kit 0515 Ordering information Product PSP Spin Stool DNA Kit PSP Spin Stool DNA Kit PSP Spin Stool DNA Kit Single components Lysis Buffer P Binding Buffer A InviAdsorb Wash Buffer Wash Buffer II Elution Buffer Product PSP Spin Stool DNA Plus Kit PSP Spin Stool DNA Plus Kit PSP Spin Stool DNA Plus Kit Single components Stool DNA Stabilizer Stool Collection Tubes with Stool DNA Stabilizer Stool Collection Tubes with Stool DNA Stabilizer Binding Buffer A Wash Buffer Wash Buffer II Elution Buffer Related products InviMag Stool DNA Mini Kit KFmL InviMag Stool DNA Mini Kit KFmL InviMag Stool DNA Mini Kit KFmL InviMag Stool DNA Mini Kit KF96 InviMag Stool DNA Mi
11. isolation and purification of viral DNA has not been evaluated Compliance with EU Directive 98 79 EC on in vitro medical devices Not for in vitro diagnostic use in countries where the EU Directive 98 79 EC on in vitro medical devices is not recognized Trademarks Invisorb PSP InviMag Eppendorf Registered marks trademarks etc used in this document even when not specifically marked as such are not to be considered unprotected by law The Invisorb technology is covered by patents and patent applications US 6 110363 US 6 043 354 US 6 037 465 EP 0880535 WO 9728171 WO 9534569 EP 0765335 DE 19506887 DE 10041825 2 WO 0034463 Invisorb PSP and InviMag are registered trademarks of STRATEC Biomedical AG The PCR process is covered by US Patents 4 683 195 and 4 683 202 and foreign equivalents owned by Hoffmann La Roche AG 2015 STRATEC Molecular all rights reserved 1 PSP Spin Stool DNA Kit 0515 PSP Spin Stool DNA Plus Kit 0515 Contents Kit components of PSP Spin Stool DNA Kit 3 Kit components of PSP Spin Stool DNA Plus Kit 4 Symbols 5 Storage 5 Quality control 5 Intended use 6 Product use limitations 6 Safety information 7 Product characteristic of PSP Spin Stool DNA Kit 8 Product characteristic of PSP Spin Stool DNA Plus Kit 9 Principle and procedure 10 Sampling and storage 10 Procedure 11 Yield and quality of total DNA 11 Important notes 12 Important points before starting a
12. liquid spillage refer to Safety Information see page 7 Do not use damaged kit components since their use may lead to poor kit performance o Always change pipet tips between liquid transfers To avoid cross contamination we recommend the use of aerosol barrier pipet tips o All centrifugation steps are carried out at room temperature When working with chemicals always wear a Suitable lab coat disposable gloves and protective goggles Discard contaminated gloves Do not combine components of different kits unless the lot numbers are identical Avoid microbial contamination of the kit reagents To minimize the risk of infections from potentially infectious material we recommend working under laminar air flow until the samples are lysed o This kit should only be used by trained personnel O OoOO 0 O Preparing reagents and buffers for the PSP Spin Stool DNA Kit and PSP Spin Stool DNA Plus Kit 1 Adjust the thermomixer to 70 C 2 Dissolve Proteinase K in ddH O 3 Warm up the needed amount of Elution Buffer to 70 C 100 200 ul Elution Buffer are needed per sample Heat heating blocks e g thermomixer to 70 C and 95 C Label the needed amount of 2 0 ml RTA Spin Filter Sets Label the needed amount of 1 5 ml Receiver Tubes per sample 1 Receiver Tube add the needed amount of ethanol to the Wash Buffer I and Il see Kit contents page 3 and 4 Dne 12 PSP Spin Stool DNA Kit 0515 PSP Sp
13. or laboratory findings To minimize irregularities in diagnostic results adequate controls for downstream applications should be used The kit is in compliance with EU Directive 98 79 EC on in vitro medical devices But it is not for in vitro diagnostic use in countries where the EU Directive 98 79 EC on in vitro medical devices is not recognized Product use limitation The kit is not validated for the isolation of genomic DNA from cultured or isolated cells from tissue swabs dried blood stains or cell free body fluid like synovial fluid and urine or the purification of RNA The application of the kit for isolation and purification of viral DNA has not been evaluated The included chemicals are only useable once Differing of starting material or flow trace may lead to inoperability therefore neither a warranty nor guarantee in this case will be given neither implied nor express The user is responsible to validate the performance of the STRATEC Molecular Product for any particular use STRATEC Molecular does not provide for validation of performance characteristics of the Product with respect to specific applications STRATEC Molecular Products may be used e g in clinical diagnostic laboratory systems conditioned upon the complete diagnostic system of the laboratory the laboratory has been validated pursuant to CLIA 88 regulations in the U S or equivalents in other countries All Products sold by STRATEC Molecular are subject
14. protocol 12 Preparing reagents and buffers 12 Equipment and reagents to be supplied by user 13 Important indications 14 Scheme of PSP Spin Stool DNA Kit 15 Protocol 1 Isolation of total DNA from up to 200 mg stool samples 16 with and without enrichment of bacterial DNA Protocol 2 Isolation of total DNA from up to 200 mg stool samples 18 from difficult to lyse bacteria Scheme of PSP Spin Stool DNA Plus Kit 20 Protocol 1 Collection of the stool sample and stabilization 21 Protocol 2 Isolation of total DNA from 1 4 ml stabilized stool 22 homogenate with and without enrichment of bacterial DNA Protocol 3 Isolation of total DNA from up to 200 mg stool samples 24 from difficult to lyse bacteria Protocol 4 Post purification of DNA containing inhibitors 26 Troubleshooting 27 Appendix 30 General notes on handling DNA 30 Determination of concentration yield and purity of DNA 30 Ordering information 31 2 PSP Spin Stool DNA Kit 0515 PSP Spin Stool DNA Plus Kit 0515 Kit components of PSP Spin Stool DNA Kit Store all kit components at room temperature RT Store diluted Proteinase K at 20 C but repeated freezing and thawing will reduced the activity dramatically Divide the Proteinase K into aliquots and store at 20 C 5 extractions 50 extractions 250 extractions Catalog No 1038100100 1038100200 1038100300 Lysis Buffer P 3x2ml 120 ml 2 x 160 Zirconia Beads Il 1 vial 2 vial 8 vials InviAdsorb 5 50 5 x 50 P
15. rpm for 2 min Discard the filtrate and the RTA Receiver Tube 24 PSP Spin Stool DNA Kit 0515 PSP Spin Stool DNA Plus Kit 0515 6 Washing Steps Put the RTA Spin Filter in a new RTA Receiver Tube Add 500 ul Wash Buffer I to the membrane of the RTA Spin Filter Close lid and centrifuge at 11 000 x g 11 000 rpm for 1 min Discard the filtrate and the RTA Receiver Tube Put the RTA Spin Filter in a new RTA Receiver Tube Add 700 ul Wash Buffer Il to the membrane of the RTA Spin Filter Close the lid and centrifuge at 11 000 x g 11 000 rpm for 1 min Discard the filtrate and put the RTA Spin Filter back to the same RTA Receiver Tube 7 Ethanol Removal To eliminate any traces of ethanol centrifuge again for 4 min at maximum speed discard the RTA Receiver Tube 8 DNA Elution Place the RTA Spin Filter into a new 1 5 ml Receiver Tube and add 100 200 ul preheated 70 C Elution Buffer Incubate for 1 min Centrifuge at 11 000 x g 11 000 rpm for 1 min to elute the DNA Finally discard the RTA Spin Filter Note The DNA can also be eluted with a lower volume of Elution Buffer depends on the expected yield of genomic DNA But pay attention that the minimum volume for the elution is 50 ul and that this volume can reduce the maximum yield If a quite large amount of DNA is expected the volume of elution can be increased Note For long term storage we recommend to keep the eluted DNA at 20 TC 25 PSP Spin Stool DNA Kit 0
16. 515 PSP Spin Stool DNA Plus Kit 0515 Additional Protocol 4 Post purification of DNA containing inhibitors Please read protocols prior the start of the preparation and complete preparing steps Important Note Stool samples are very heterologous depending on nutritation of the producer and source of the stool In some cases inhibitors for downstream reactions might occur in the eluted DNA In this case the following post purifying protocol may help 1 Eluate adjustment Adjust your eluate to at least 100 ul for respective dilution take water 2 Sephadex G50 Slurry Make slurry of Sephadex G50 by adding water to Sephadex G50 powder und soaking until the slurry is reaching its final extension This is dependent on the amount you are producing it shouldn t take more than 30 minutes 3 Adsorbtion of inhibitors Add 1 3 of your eluate volume of slurry to the eluate Incubate for 30 minutes under continuous shaking at room temperature RT 4 Removal of slurry Centrifuge the mixture at 11 000 x g 11 000 rpm for 1 min Take the supernatant and transfer it to a new reaction tube it contains you purified DNA This purification may be repeated once but remember that you will loose 25 of yield during every purification step 26 PSP Spin Stool DNA Kit 0515 PSP Spin Stool DNA Plus Kit 0515 Troubleshooting Problem Comments and suggestions Clogged RTA Spin Filter insufficient lysis and or too much starting ma
17. If a quite large amount of DNA is expected the volume of elution can be increased Note For long term storage we recommend to keep the eluted DNA at 20 TC 17 PSP Spin Stool DNA Kit 0515 PSP Spin Stool DNA Plus Kit 0515 Protocol 2 Isolation of total DNA from up to 200 mg stool samples from difficult to lyse bacteria Please read protocols prior the start of the preparation and complete preparing steps Attention Please be aware that you have to prepare the Binding Buffer A see instruction page 13 Important Note To lyse some special bacteria completely like Mycobacteria paratuberculosis or Chlamydia a special treatment is necessary Heat heating blocks e g thermomixer to 70 C and 95 Prepare a container with crushed ice Preheat the Elution Buffer to 70 e g transfer the needed volume into a tube and place it at the appropriate temperature into a thermomixer 1 Sample homogenization and prelysis Weigh 200 mg of stool sample fresh or frozen into a 2 0 ml Safe Lock Tube and add 1 2 ml Lysis Buffer P to each stool sample Vortex vigorously for 1 min Important If the sample is liquid pipet 200 ul into the 2 0 ml Safe Lock Tube Cut the end of the pipet tip to make pipetting easier Incubate the homogenized sample for 10 min at 95 C in a thermomixer under continuous shaking at 900 rpm Incubate the sample on ice for 3 minutes Add 5 Zirconia Beads II to the homogenate Put the sample back to the
18. after storage or directly after collection into the 2 0 ml Safe Lock Tube Incubate the homogenized sample for 10 min at 95 C in a thermomixer under continuous shaking at 900 rpm Incubate the sample on ice for 3 minutes and put the sample back to the 95 C thermo block Incubate for further 3 min at 95 C Add 5 Zirconia Beads II to the homogenate and vortex for 2 min at RT Centrifuge the sample at 11 000 x g 11 000 rpm for 1 min to pellet solid stool particles 2 Removal of PCR Inhibitiors Transfer the supernatant into an InviAdsorb Tube and vortex vigorously for 15 sec Incubate the suspension for 1 min at room temperature Centrifuge the sample at full speed for 3 min 3 Second Sample Cleanup Transfer the supernatant completely into a new 1 5 ml Receiver Tube Discard the pellet Centrifuge the sample again at full soeed for 3 min 4 Proteinase K digestion Transfer 25 ul Proteinase K into a new 2 0 ml Safe Lock Tube and pipet 800 ul of the supernatant from step 3 to the 1 5 ml Receiver Tube containing Proteinase K mix shortly by vortexing and incubate the sample for 10 min at 70 C in a thermomixer under continuous shaking at 900 rpm 5 Binding of the DNA Add 400 ul of Binding Buffer A to the lysate and mix shortly by vortexing or by pipetting up and down several times Transfer the mixture in two steps onto the membrane of the RTA Spin Filter Incubate for 1 min at room temperature and centrifuge at 11 000 x g 11 000
19. an be used This can be removed by RNase digestion The carrier should be added to the lysis buffer before preparation or to the stabilization buffer never add to the stool directly Invisorb RTA Spin filter can also purify low amounts of RNA besides DNA For the elimination of RNA if necessary add 20 ul RNase A 10 mg ml before adding the Binding Buffer A Vortex briefly and incubate the sample at room temperature for 5 minutes Then go on as described in the protocol Elution of DNA O For downstream applications that require small starting volumes a more concentrated eluate may increase assay sensitivity The elution can be done by using a lower volume of Elution Buffer down to 60 ul This may result in a higher concentration of DNA But lower volumes of Elution Buffer will decrease the yield of DNA The final volume of eluate recovered may be up to 5 ul less than the volume of elution buffer applied to the spin filter If low concentrated TRIS buffer affects sensitive downstream applications use distilled sterile water for elution However ensure that the pH of the water is at least 7 0 deionized water from certain sources can be acidic DNA stored in water is subjected to degradation by acid hydrolysis Eluting twice with each 100 ul Elution Buffer is also possible and gives slightly higher yield of DNA Handling of the RTA Spin Filter Set Due to the sensitivity of DNA amplification technologies the following precautions
20. contains InviAdsorb a reagent that efficiently adsorbs these components at the beginning of the purification process and additionally optimized essential washing conditions for the final removal of the last traces of all potent inhibitors See more to the procedure at page 11 and19 Due to the high purity the isolated DNA is suitable for a broad panel of downstream applications see below or can be stored at 20 C for later use PCR applications RFLP analysis Hybridization Genetic typing Pathogen typing Mutation analysis Paternity testing OoO0O0O000 0 To purify high molecular weight total DNA using magnetic beads STRATEC Molecular offers different kits in single sample format as well as 15 well or in 96 format the InviMag Stool DNA Mini Kit for use on the InviMag Rack the InviMag Stool DNA Mini Kit KFmL and KF96 for use on the KingFisher workstations The PCR method i is covered by U S Patents 4 683 195 and 4 683 202 owned by Hoffmann LaRoche Inc The purchase of the PSP Spin Stool DNA Plus Kit cannot be construed as an authorization or implicit licence to practice PCR under any patents held by Hoffmann LaRoche Inc 9 PSP Spin Stool DNA Kit 0515 PSP Spin Stool DNA Plus Kit 0515 Principle and procedure The PSP Spin Stool DNA Kit procedure comprises following steps lysis of sample removal of PCR inhibitors protein digestion binding the nucleic acids to the membrane of a spin column washing of the sp
21. e and mix shortly by vortexing or by pipetting up and down several times Transfer the mixture completely onto the membrane of the RTA Spin Filter Incubate for 1 min at room temperature and centrifuge at 11 000 x g 11 000 rpm for 2 min Discard the filtrate and the RTA Receiver Tube 6 Washing steps Put the RTA Spin Filter in a new RTA Receiver Tube Add 500 ul Wash Buffer I to the membrane of the RTA Spin Filter Close the lid and centrifuge at 11 000 x g 11 000 rpm for 1 min Discard the filtrate and the RTA Receiver Tube Put the RTA Spin Filter in a new RTA Receiver Tube Add 700 ul Wash Buffer Il to the membrane of the RTA Spin Filter Close the lid and centrifuge and centrifuge at 11 000 x g 11 000 rpm for 1 min Discard the filtrate and put the RTA Spin Filter back to the same RTA Receiver Tube 7 Ethanol removal To eliminate any traces of ethanol centrifuge again for 4 min at maximum speed discard the RTA Receiver Tube 8 DNA Elution Place the RTA Spin Filter into a new 1 5 ml Receiver Tube and add 100 200 ul preheated 70 C Elution Buffer to the sample Incubate for 1 min at RT Centrifuge at 11 000 x g 11 000 rpm for 1 min to elute the DNA Finally discard the RTA Spin Filter Note The DNA can also be eluted with a lower volume of Elution Buffer depends on the expected yield of genomic DNA But pay attention that the minimum volume for the elution is 50 ul and that this volume can reduce the maximum yield
22. eded volume into a tube and place it at the appropriate temperature into a thermomixer 1 Sample Homogenization and Prelysis Transfer 1 4 ml of the collected and well homogenized stool sample Stool DNA Stabilizer with stool specimen after storage or directly after collection into the 2 0 ml Safe Lock Tube Centrifuge the sample at 11 000 x g 11 000 rpm for 1 min to pellet solid stool particles This will lead to a reduced amount of extracted total DNA but is not influencing the amount of human DNA For an enrichment of bacterial DNA Incubate the sample for 10 min at 95 C in a thermomixer under continuously shaking at 900 rpm Add 5 Zirconia Beads II to the homogenate and vortex for 2 min at RT Centrifuge the sample at 11 000 x g 11 000 rpm for 1 min to pellet solid stool particles and beads Important Note The incubation step at 95 C will lead to maximize the amount of bacterial DNA because of a very efficient destruction of the cell wall of e g gram bacteria For an enrichment of host DNA don t perform this high temperature step 2 Removal of PCR Inhibitiors Transfer the supernatant into an InviAdsorb Tube and vortex vigorously for 15 sec Incubate the suspension for 1 min at room temperature Centrifuge the sample at full speed for 3 min 3 Second Sample Cleanup Transfer the supernatant completely into a new 1 5 ml Receiver Tube Discard the pellet Centrifuge the sample again at full soeed for 3 min 4 Prot
23. einase K digestion Transfer 25 ul Proteinase K into a new 2 0 ml Safe Lock Tube and pipet 800 ul of the supernatant from step 3 to the 1 5 ml Receiver Tube containing Proteinase K mix shortly by vortexing and incubate the sample for 10 min at 70 C in a thermomixer under continuous shaking at 900 rpm 22 PSP Spin Stool DNA Kit 0515 PSP Spin Stool DNA Plus Kit 0515 5 Binding of the DNA Add 400 ul of Binding Buffer A to the lysate and mix shortly by vortexing or by pipetting up and down several times Transfer the mixture in two steps onto the membrane of the RTA Spin Filter Incubate for 1 min at room temperature and centrifuge at 11 000 x g 11 000 rpm for 2 min Discard the filtrate and the RTA Receiver Tube 6 Washing Steps Put the RTA Spin Filter in a new RTA Receiver Tube Add 500 ul Wash Buffer I to the membrane of the RTA Spin Filter Close the lid and centrifuge at 11 000 x g 11 000 rpm for 1 min Discard the filtrate and the RTA Receiver Tube Put the RTA Spin Filter in a new RTA Receiver Tube Add 700 ul Wash Buffer Il to the membrane of the RTA Spin Filter Close the lid and centrifuge at 11 000 x g 11 000 rpm for 1 min Discard the filtrate and put the RTA Spin Filter back to the same RTA Receiver Tube 7 Ethanol Removal To eliminate any traces of ethanol centrifuge again for 4 min at maximum speed discard the RTA Receiver Tube 8 DNA Elution Place the RTA Spin Filter into a new 1 5 ml Receiver Tube and
24. ent mixing with the InviAdsorb matrix insufficient mixing with Lysis Buffer P decreased proteinase activity no Binding Buffer A added to the lysate Wash Buffer and Wash Buffer Il prepared incorrectly repeat the DNA purification procedure with a new sample be sure to mix the sample and InviAdsorb matrix until the sample is thoroughly homogenized repeat the procedure with a new sample be sure to mix the sample and Lysis Buffer P immediately and thoroughly by pulse vortexing repeat the DNA purification procedure with a new sample and with Proteinase K for difficult cases use double volume Proteinase K repeat the purification procedure with a sample check that Wash Buffer I and Wash Buffer II were diluted with 96 100 ethanol do not use denatured alcohol which contains other substances such as methanol or methylethylketone repeat the purification procedure with a new sample 27 PSP Spin Stool DNA Kit 0515 PSP Spin Stool DNA Plus Kit 0515 A260 A280 ratio for purified nucleic acids is low Wash Buffer I and Wash Buffer Il used in the wrong order protein contamination ensure that Wash Buffer and Wash Buffer Il are used in the correct order in the protocol repeat washing step with Wash Buffer in the repeated preparation DNA does not perform well in downstream applications BSA not added to PCR mixture too much DNA used in downstream reaction nonspecific bands in inefficient lysis of tar
25. ently removed during the following three wash steps and highly purified DNA is eluted in Elution Buffer or water This manual contains 4 protocols Sampling and storage of starting material PSP Spin Stool DNA Kit The collected fresh stool sample can be stored at ambient temperature for at least 1 2 hours at RT but the high content of DNases realize quickly a DNA digestion and degradation The sample should be quickly added to the Lysis Buffer P or can be stored frozen at 20 C for weeks PSP Spin Stool DNA Plus Kit The storage of fresh samples in Stool DNA Stabilizer provided in the Stool Collection Tubes allow storage at RT for about 3 days The storage of fresh samples in Stool DNA Stabilizer will lead to less degraded DNA and a better yield of bacterial pathogens with difficult to lyse cell walls Storage time below 3 days has no influence on the quality or the amount of host cell DNA The collected sample in Stool DNA Stabilizer can also be used immediately for the isolation of DNA after collection The collected sample can be refrigerated at 20 C immediately after collection or after storage at ambient temperature for a later use for example for a second DNA isolation STRATEC Molecular will be released of its responsibilities if other sample materials than described in the Intended Use are processed or if the sample preparation protocols are changed or modified 10 PSP Spin Stool DNA Kit 0515 PSP Spin Stool DNA Plus
26. er Tube pipet 700 ul Wash Buffer Il onto the RTA Spin Filter centrifuge at 11 000 x g 11 000 rpm for 1 min discard the flow through and reuse the RTA Receiver Tube to eliminate any traces of ethanol centrifuge again for 4 min at maximum speed discard the RTA Receiver Tube transfer the RTA Spin Filter into a new 1 5 ml Receiver Tube pipet 100 200 ul of Elution Buffer preheated to 70 C directly onto the center of the membrane of the RTA Spin Filter incubate for 1 min at RT centrifuge at 11 000 x g 11 000 rpm for 1 min discard the RTA Spin Filter place the eluted total DNA immediately in a refrigerator or store it at 20 C 20 PSP Spin Stool DNA Kit 0515 PSP Spin Stool DNA Plus Kit 0515 Instructions The following notes are valid for all protocols Note The centrifugation steps were made with the Centrifuge 5415 D from Eppendorf The indicated rpm amounts are referring to this centrifuge Protocol 1 Collection of the stool sample and stabilization Please read protocols prior the start of the preparation and complete preparing steps Note The Stool Collection Tubes contains 8 ml of Stool DNA Stabilizer That is a new developed buffer formulation which enables the prelysis and stabilization of the DNA for at least 3 days at ambient temperature The Stool DNA Stabilizer is very successful even if bacterial pathogens should be detected which are difficult to lyse because of the structure of their cell walls 1
27. es with and without enrichment of bacterial DNA page 16 See also protocol Post Purification see page 25 ensure that the Wash Buffer I and Il are used in the correct order in the protocol add 400 ul Lysis Buffer P and 200 ul Binding Buffer A to the eluate and continue with step 5 of Protocol Isolation of total DNA from up to 400 mg stool samples with and without enrichment of bacterial DNA page 15 insufficient mixing with Lysis Buffer P or Stool DNA Stabilizer reduced sensitivity of amplification reaction repeat the purification with other aliquots determine the maximum volume of eluate amplification reaction suitable for your amplification reaction Reduce or increase the volume of eluate added to the reaction optimize your amplification system e g by changing template volume 28 PSP Spin Stool DNA Kit 0515 PSP Spin Stool DNA Plus Kit 0515 Little or no supernatant visible after initial centrifugation step insufficient centrifugal force increase the centrifugation time proportionately if your centrifuge cannot provide 13 400 x g 12 000 rpm e g instead of centrifuging for 1 minutes at 13 400 x g centrifuge for 3 minutes at 10 000 x g Little or no supernatant visible after centrifugation step with InviAdsorb matrix insufficient centrifugal force with some samples centrifugation to precipitate the InviAsorb matrix may result in a pellet that is not sufficiently compac
28. get cells not enough DNA in eluate inhibitory substances in preparation residual Wash Buffer in the eluate when using eluates in PCR for maximum PCR robustness add BSA or Solution to a final concentration of 0 1 ug ul to the PCR mixture the PSP Spin Stool DNA Kit and PSP Spin Stool DNA Plus Kit purifies total DNA which could originate from many different organisms present in the original stool sample e g human animal plant bacterial If the amount of total DNA is too high PCR could be inhibited by excess total DNA Reduce the amount of eluate used in the downstream reaction if possible it is likely that only a low quantity of target downstream PCR DNA is present in stool sample eluates together with high amounts of background DNA the amount of target DNA in the eluate may be low if the target cells are difficult to lyse as is the case with some bacteria and parasites In future preparations prolong incubation time of the sample at 95 C and or add zirconia beads to the stool samples lysis mixture see PSP Spin Stool DNA Kit protocol 2 page 17 check Low amount or no DNA of extracted DNA for possible reasons see A260 A280 ratio for purified nucleic acids is low for possible reasons Bring the eluate volume to 200 ul add to the supernatant 400 ul Lysis Buffer P and mix all with 200 ul Binding Buffer A Repeat the protocol 1 from step 5 of Isolation of total DNA from up to 400 mg stool sampl
29. he sample at 11 000 x g 11 000 rpm for 1 min to pellet solid stool particles For an enrichment of bacterial DNA Incubate the sample for 10 min at 95 C in a thermomixer under continuously shaking at 900 rpm Add 5 Zirconia Beads II to the homogenate and vortex for 2 min at RT Centrifuge the sample at 11 000 x g 11 000 rpm for 1 min to pellet solid stool particles and beads Important The incubation step at 95 C will maximize the yield of bacterial DNA because of a very efficient disruption of the cell wall of e g gram positive bacteria For an enrichment of host DNA don t perform this high temperature step 2 Removal of PCR inhibitiors Transfer the supernatant into an InviAdsorb Tube and vortex vigorously for 15 sec Incubate the suspension for 1 min at room temperature Centrifuge the sample at full speed for 3 min 16 PSP Spin Stool DNA Kit 0515 PSP Spin Stool DNA Plus Kit 0515 3 Second sample cleanup Transfer the supernatant completely into a new 1 5 ml Receiver Tube Discard the pellet Centrifuge the sample again at full soeed for 3 min 4 Proteinase K digestion Transfer 25 ul Proteinase K into a new 2 0 ml Safe Lock Tube and pipet 400 ul of the supernatant from step 3 to the 1 5 ml Receiver Tube containing Proteinase K Mix shortly by vortexing and incubate the sample for 10 min at 70 C in a thermomixer under continuous shaking at 900 rpm 5 Binding of the DNA Add 200 ul Binding Buffer A to the lysat
30. id and centrifuge at 11 000 x g 11 000 rpm for 1 min Discard the filtrate and the RTA Receiver Tube Put the RTA Spin Filter in a new RTA Receiver Tube Add 700 ul Wash Buffer Il to the membrane of the RTA Spin Filter Close the Close the lid and centrifuge at 11 000 x g 11 000 rpm for 1 min Discard the filtrate and put the RTA Spin Filter back to the same RTA Receiver Tube 7 Ethanol Removal To eliminate any traces of ethanol centrifuge again for 4 min at maximum speed discard the RTA Receiver Tube 8 DNA Elution Place the RTA Spin Filter into a new 1 5 ml Receiver Tube and add 100 200 ul preheated 70 C Elution Buffer Incubate for 1 min Centrifuge at 11 000 x g 11 000 rpm for 1 min to elute the DNA Finally discard the RTA Spin Filter Note The DNA can also be eluted with a lower volume of Elution Buffer depends on the expected yield of genomic DNA But pay attention that the minimum volume for the elution is 50 ul and that this volume can reduce the maximum yield If a quite large amount of DNA is expected the volume of elution can be increased Note For long term storage we recommend to keep the eluted DNA at 20 TC 19 PSP Spin Stool DNA Kit 0515 PSP Spin Stool DNA Plus Kit 0515 Scheme of the PSP Spin Stool DNA Plus Kit Please read protocols prior the start of the preparation carefully collect a spoon of the stool sample transfer stool sample into the Stool Collection Tube close the tube
31. in Stool DNA Plus Kit 0515 5 total DNA extractions add 250 ul ddH20 to Proteinase K mix thoroughly until completely dissolving and store the tube at 20 C Binding Buffer A Wash Buffer I and Il are ready to use 50 total DNA extractions PSP Spin Stool DNA Kit add 11 ml 99 7 Isopropanol to the Binding Buffer A Mix by intensive shaking by inverting for 1 min PSP Spin Stool DNA Plus Kit add 21 ml 99 7 Isopropanol to the Binding Buffer A Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times add 1 5 ml ddH20 to Proteinase K mix thoroughly until completely dissolving and store the tube at 20 C add 30 ml 96 100 ethanol to the bottle Wash Buffer I add 42 ml 96 100 ethanol to each bottle Wash Buffer Il mix thoroughly and always keep the bottle firmly closed 250 total DNA extractions PSP Spin Stool DNA Kit add 21 ml 99 7 Isopropanol to each Binding Buffer A Mix by intensive shaking by inverting for 1 min PSP Spin Stool DNA Plus Kit add 84 ml 99 7 lsopropanol to the Binding Buffer A Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times add 1 5 ml ddH2O to Proteinase K mix thoroughly until completely dissolving and store the tube at 20 C add 80 ml 96 100 ethanol to each bottle Wash Buffer I add 105 ml 96 100 ethanol to each bottle Wash Buffer II mix thoroughly and always keep the bottle firmly closed
32. in column and hereby elimination of contaminants and ethanol o elution of the nucleic acids oo0oo0o0o0 After homogenization of the sample in the Lysis Buffer P which inactivates DNases the human cells and the bacterial cell wall will be lysed differently depending on the temperature profile The lysate will be mixed with InviAdsorb and most of the PCR inhibiting components will be removed followed by a protein digestion After lysis the DNA binds to the membrane contaminations and enzyme inhibitors are efficiently removed during the following three washing steps and highly purified DNA is eluted in Elution Buffer or water The PSP Spin Stool DNA Plus Kit procedure comprises some additional steps likes o sample collection o DNA stabilization in the sample o followed by all steps from the PSP Spin Stool DNA Kit procedure After sample collection and homogenization of the sample in the Stool Collection Tube containing the DNA stabilizing Lysis Buffer which inactivates DNases the human and bacterial cells will be lysed differently depending from the temperature profile The DNA is stable for more than 3 days and can be transported to the lab without degradation prelysing bacteria and stopping further bacterial grow The lysate will be mixed later with InviAdsorb and the most PCR inhibiting components will be removed followed by a protein digestion After lysis the DNA binds to the membrane contaminations and enzyme inhibitors are effici
33. mine the suitability of the Product for its particular use Should any Product fail to perform the applications as described in the manual STRATEC Molecular will check the lot and if STRATEC Molecular investigates a problem in the lot STRATEC Molecular will replace the Product free of charge STRATEC Molecular reserves the right to change alter or modify any Product to enhance its performance and design at any time In accordance with STRATEC Molecular s ISO 9001 2000 and ISO EN 13485 certified Quality Management System the performance of all components of the PSP Spin Stool DNA Kit and the PSP Spin Stool DNA Plus Kit have been tested separately against predetermined specifications routinely on lot to lot to ensure consistent product quality If you have any questions or problems regarding any aspects of PSP Spin Stool DNA Kit and the PSP Spin Stool DNA Plus Kit or other STRATEC Molecular products please do not hesitate to contact us A copy of STRATEC Molecular s terms and conditions can be obtained upon request or are presented at the STRATEC Molecular webpage For technical support or further information please contact from Germany 49 0 30 9489 2901 2910 from abroad 49 0 30 9489 2907 or contact your local distributor 5 PSP Spin Stool DNA Kit 0515 PSP Spin Stool DNA Plus Kit 0515 Intended use The PSP Spin Stool DNA Kit has been designed for fast and efficient purification of genomic and microbial DNA from up
34. mixer at 900 rpm add 200 ul Binding Buffer A follow preparing instructions to the lysate mix shortly by vortexing or pipetting up and down transfer the whole mixture to the RTA Spin Filter incubate for 1 min at RT centrifuge at 11 000 x g 11 000 rpm for 2 min discard the filtrate and the RTA Receiver Tube transfer the RTA Spin Filter in a new RTA Receiver Tube pipet 500 ul Wash Buffer I onto the RTA Spin Filter centrifuge at 11 000 x g 11 000 rpm for 1 min discard the flow through and the RTA Receiver Tube put the RTA Spin Filter in a new RTA Receiver Tube pipet 700 ul Wash Buffer Il onto the RTA Spin Filter centrifuge at 11 000 x g 11 000 rpm for 1 min discard the flow through and reuse the RTA Receiver Tube to eliminate any traces of ethanol centrifuge again for 4 min at maximum speed discard the RTA Receiver Tube transfer the RTA Spin Filter into a new 1 5 ml Receiver Tube pipet 100 200 ul of Elution Buffer preheated to 70 C directly onto the center of the membrane of the RTA Spin Filter incubate for 1 min at RT centrifuge at 11 000 x g 11 000 rpm for 1 min discard the RTA Spin Filter place the eluted total DNA immediately on ice and store at 20 C 15 PSP Spin Stool DNA Kit 0515 PSP Spin Stool DNA Plus Kit 0515 Instructions The following notes are valid for all protocols Note The centrifugation steps were made with the Centrifuge 5415 D from Eppendorf The indicated refers to
35. ni Kit KF96 Package size 5 extractions 50 extractions 250 extractions 30 ml 9 ml for 10 purifications 30 ml 18 ml 15 ml Package size 5 purifications 50 purifications 250 purifications 250 ml 50 tubes 250 tubes 9 ml 30 ml 18 ml 15 ml 15 purifications 75 purifications 300 purifications 1 x 96 purifications 5 x 96 purifications Possible suppliers for lsopropanol Carl Roth 2 Propanol Rotipuran gt 99 7 p a ACS ISO Order no 6752 Applichem 2 Propanol f r die Molekularbiologie Order no A3928 31 Catalogue No 1038100100 1038100200 1038100300 1038101200 1038102800 1038107600 1038103300 1038103400 1038104000 Catalogue No 1038110100 1038110200 1038110300 1038111100 1038111200 1038111300 1038112800 1038113300 1038113400 1038114000 2438110100 2438110200 2438110400 7438300100 7438300200 Sigma 2 Propanol Order no 59304 1L F PSP Spin Stool DNA Kit 0515 PSP Spin Stool DNA Plus Kit 0515 Sstratecee molecular STRATEC Molecular GmbH Robert Rossle Str 10 13125 Berlin Germany Phone 49 30 94 89 29 01 Fax 49 30 94 89 29 09 E mail info berlin stratec com www stratec com 1A3f0102 05 2015
36. nvisorb membrane Removing residual contaminants DNA bound to the Invisorb membrane is washed in three centrifugation steps Contaminants are efficiently and completely removed using Wash Buffer and Il while the nucleic acids remain bound to the membrane Elution The nucleic acids are eluted in low salt buffer from the membrane using 100 200 ul Elution Buffer The eluted nucleic acids are ready to use in different subsequent tests Yield and quality of total DNA The amount of purified DNA in the PSP Spin Stool DNA Kit procedure from feces depends on the health status of the donor bacteria content sample source transport storage and age A typical yield is 10 80 ug a typical DNA concentration is 50 300 ng ul Yield and quality of isolated genomic DNA is suitable for any molecular diagnostic detection system The diagnostic tests should be performed according to manufacturers specifications 11 PSP Spin Stool DNA Kit 0515 PSP Spin Stool DNA Plus Kit 0515 Important notes Important points before starting a protocol Immediately upon receipt of the Product inspect the Product and its components as well as the package for any apparent damages correct quantities and quality If there are any unconformities you have to notify STRATEC Molecular in writing with immediate effect upon inspection thereof If buffer bottles are damaged contact the STRATEC Molecular Technical Services or your local distributor In case of
37. of genomic DNA from cells or tissues use either fresh samples or samples that have been quickly frozen in liquid nitrogen and stored at 70 C This procedure minimizes degradation of crude DNA by limiting the activity of endogenous nucleases Storage of DNA Store genomic DNA at 2 to 8 C Storing genomic DNA at 15 to 25 C can cause shearing of DNA particularly if the DNA is exposed to repeated freeze thaw cycles Plasmid DNA and other small circular DNAs can be stored at 2 to 8 C or at 15 to 25 C Drying dissolving and pipetting DNA Avoid overdrying genomic DNA after ethanol precipitation It is better to let it air dry than to use a vacuum although vacuum drying can be used with caution Plasmid DNA and other small circular DNAs can be vacuum dried To help dissolve the DNA carefully invert the tubes several times after adding buffer and tap the tube gently on the side Alternatively let the DNA stand in buffer overnight at 2 to 8 C Minimize vortexing of genomic DNA since this can cause shearing Avoid vigorous pipetting Pipetting genomic DNA through small tip openings causes shearing or nicking One way to decrease shearing of genomic DNA is to use special tips that have wide openings designed for pipetting genomic DNA Regular pipette tips pose no problem for plasmid DNA and other small fragments Determination of concentration yield and purity of DNA Determination of concentration yield and purity DNA yields are
38. ol DNA Kit max 200 mg fecal sample up to 50 ug depends on about 45 min A260 A 280 starting material incl lysis time 1 4 1 8 The PSP Spin Stool DNA Kit allows rapid and efficient isolation of high quality DNA from up to 200 mg of fresh or frozen stool sample Stool samples typically contain many compounds that can degrade DNA and inhibit downstream enzymatic reactions To ensure the removal of these contaminants the PSP Spin Stool DNA Kit contains tubes with InviAdsorb and optimized essential washing conditions to remove all potent inhibitors very efficiently So the simple PSP Spin procedure yields pure DNA ready to use in less than 1h A rigorous prelysis step using Zirconia Beads II with optimized prelysis buffer under high temperatures is followed by a preincubation of the sample with InviAdsorb to remove PCR inhibitors Undissolved particles and PCR inhibitors bound to InviAdsorb are removed by a centrifugation step The following Proteinase K digestion ensures high yields also from gram positive bacteria Stool contains a range of DNA e g host DNA from colon epithelial cells parasite DNA bacterial DNA DNA from food or DNA from gastrointestinal pathogens The choise of different lysis conditions allows the enrichment or a reduction of the content of bacterial DNA in the total DNA All impurities are removed very efficiently during washing steps and the purified DNA is eluted directly in a low salt buffer No phenol chlorofo
39. rm extraction or ethanol precipitation is necessary The kit provides reproducible recovery rates of highly purified DNA ready to use in any downstream application The isolated DNA can be stored at 20 C for later use Due to the high purity the isolated total DNA is suitable for a broad panel of downstream applications see below or can be stored at 80 C for subsequent use PCR applications RFLP analysis Hybridization Genetic typing Pathogen typing Mutation analysis oo0oo0o0o0o0 No toxic or hazardous chemicals like phenol chloroform or B Mercaptoethanol are used Traditional time killing procedures can be replaced using the PSP Spin Stool DNA Kit To increase robustness of PCR assays using DNA isolated from stool samples the addition of BSA to a final concentration of 0 1 ug ul to the PCR mixtures is recommended In DNA eluates from feces the ratio of target DNA to background DNA is often very low The PCR method is covered by U S Patents 4 683 195 and 4 683 202 owned by Hoffmann LaRoche Inc The purchase of the PSP Spin Stool DNA Kit cannot be construed as an authorization or implicit licence to practice PCR under any patents held by Hoffmann LaRoche Inc 8 PSP Spin Stool DNA Kit 0515 PSP Spin Stool DNA Plus Kit 0515 Product characteristic of the PSP Spin Stool DNA Plus Kit 1 4 ml Stool DNA Stabilizer up to 50 ug depends on about 45 min A260 A 280 with stool homogenate starting material incl ly
40. ropanol to each Binding Buffer A Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times Add 1 5 ml ddH20 to each tube Proteinase K mix thoroughly until completely dissolving and store the tube at 20 C Add 80 ml of 96 100 ethanol to the bottle Wash Buffer and always keep the bottle firmly closed Add 105 ml of 96 100 ethanol to each bottle Wash Buffer Il mix thoroughly and always keep the bottle firmly closed Incubate the needed amount of Elution Buffer at 70 C ina thermomixer thermomixer 3 PSP Spin Stool DNA Kit 0515 PSP Spin Stool DNA Plus Kit 0515 Kit components of PSP Spin Stool DNA Plus Kit Store all kit components at room temperature RT Store diluted Proteinase K at 20 C but repeated freezing and thawing will reduced the activity dramatically Divide the Proteinase K into aliquots and store at 20 C 5 extractions 50 extractions 250 extractions Catalog No 1038110100 1038110200 1038110300 Stool Collection Tubes with Stool DNA Stabilizer 9 2x29 axe InviAdsorb 5 50 5 x50 Zirconia Beads Il 1 vial 2 vial 8 vials Proteinase K for 250 ul for 1 5 ml for5x 1 5 ml working solution working solution working solution Binding Buffer A 3x1ml ready to use 9 ml final volume 30 ml 36 ml final volume 120 ml Wash Buffer 15ml ready to use 30 ml final volume 60 ml 80 ml final vol
41. roteinase K for 250 ul for 1 5 ml for 5 x 1 5 ml working solution working solution working solution Binding Buffer A 2x1ml ready to use 4 ml final volume 15 ml 2x9ml final volume 2 x 30 ml Wash Buffer 15 ml ready to use 30 ml final volume 60 ml 80 ml final volume 160 ml Wash Buffer Il 15 ml ready to use 18 ml final volume 60 ml 2x 45 ml final volume 2 x 150 ml Elution Buffer 2 ml 15 ml 60 ml 2 0 ml Safe Lock Tubes 10 2x 50 10 x 50 RTA Spin Filter Set 5 50 5 x 50 RTA Receiver Tubes 2x5 2x50 10 x 50 1 5 ml Receiver Tubes 2x5 2x50 10x 50 Manual 1 1 Add 11 ml 99 7 Isopropanol Initial steps Add 250 ul ddH20 to Proteinase K mix thoroughly until completely dissolving and store the tube at 20 C Incubate the needed amount of Elution Buffer at 70 C in a thermomixer to the Binding Buffer A Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times Add 1 5 ml ddH20 to Proteinase K mix thoroughly until completely dissolving and store the tube at 20 C Add 30 ml of 96 100 ethanol to the bottle Wash Buffer mix thoroughly and always keep the bottle firmly closed Add 42 ml of 96 100 ethanol to the bottle Wash Buffer Il mix thoroughly and always keep the bottle firmly closed Incubate the needed amount of Elution Buffer at 70 C ina Add 21 ml 99 7 Isop
42. sis time 1 4 1 8 The PSP Spin Stool DNA Plus Kit combines the collection of stool samples the storage and stabilization of the stool specimen without any degradation of the DNA with a very efficient and fast isolation of high quality total DNA The PSP Spin Stool DNA Plus Kit can be used for isolation of DNA from pathogenic microorganism as well as for isolation of DNA from host organism The PSP Spin Stool DNA Plus Kit uses a stabilizing reagent the Stool DNA Stabilizer which enables the storage of the stool samples after collection without cooling under ambient temperature for at least 3 days The system combines the use of the Stool Collection Tubes prefilled with the Stool DNA Stabilizer for collection storage and stabilization of the stool specimen without any degradation of the DNA during transportation and the prelysis of bacteria with a very efficient and fast isolation of high quality DNA from stool sample In addition to the inactivation of DNases the Stool DNA Stabilizer preserves the microorganism titer Furthermore the Stool DNA Stabilizer enables prelysis of gram positive or negative bacteria For the DNA extraction process only a small amount of the total volume will be used the residual sample can be used for further extractions or a long term storage at 20 C Stool samples typically contain many components that can degrade DNA and inhibit down stream enzymatic reactions Therefore the PSP Spin Stool DNA Plus Kit
43. t in these cases it is recommended to increase the centrifugation time for precipitation of InviAdsorb matrix to 6 minutes Precipitate after addition of Binding Buffer A General handling lysate not completely passed through silica membrane cross contamination between samples in most cases this effect comes frm big amounts of DNA in the sample Don t remove this precipitate and follow strictly the protocol centrifuge for 1 minute at full soeed or until all the lysate has passed through the membrane to avoid cross contamination when handling RTA Spin columns read Handling of RTA Spin Filter on page 12 repeat the purification procedure with new samples 29 PSP Spin Stool DNA Kit 0515 PSP Spin Stool DNA Plus Kit 0515 Appendix General notes on handling DNA Nature of DNA The length and delicate physical nature of DNA require careful handling to avoid damage due to shearing and enzymatic degradation Other conditions that affect the integrity and stability of DNA include acidic and alkaline environments high temperature and UV irradiation Careful isolation and handling of high molecular weight DNA is necessary to ensure it will function well in various downstream applications Damaged DNA could perform poorly in applications such as genomic Southern blotting long template PCR and construction of cosmid libraries Handling fresh and stored material before the extraction of DNA For the isolation
44. terial increase lysis time increase centrifugation speed reduce amount of starting material Low amount or no DNA of extracted DNA sample stored incorrectly insufficient homogenization of stool sample in Lysis Buffer P or in Stool DNA Stabilizer insufficient lysis insufficient mixing of the sample with Binding Buffer A no alcohol added to the Wash Buffer I and Il DNA not eluted efficiently sample should be stored at 4 C or 20 C repeat the DNA purification procedure with a new sample Be sure to mix the sample and Lysis Buffer P or in Stool DNA Stabilizer until the sample is thoroughly homogenized use Zirconia Beads II and vortex for homogenization increase lysis time reduce amount of starting material overloading of Spin Filter reduces yield mix sample sufficient by pipetting up and down with Binding Buffer A prior to transfer the sample onto the RTA Spin Filter membrane check that Wash Buffer and Wash Buffer II concentrates were diluted with correct volume of 96 100 ethanol repeat the purification procedure with a new sample to increase elution efficiency pipet the preheated Elution Buffer onto the center of the RTA Spin Filter and incubate the column for 5 minutes at room temperature before centrifugation do the elution steps twice take higher volume of Elution Buffer A260 A280 ratio for purified nucleic acids is low inefficient elimination of inhibitory substances due to insuffici
45. this centrifuge Protocol 1 Isolation of total DNA from up to 200 mg stool samples with and without enrichment of bacterial DNA Please read protocols prior the start of the preparation and complete preparing steps Attention Please be aware that you have to prepare the Binding Buffer A see instruction page 13 Important Note Please note that the majority of extracted DNA from stool samples is of bacterial origin Heat heating blocks e g thermomixer to 70 C and 95 CT Preheat the Elution Buffer to 70 e g transfer the needed volume into a tube and place it at the appropriate temperature into a thermomixer 1 Sample homogenization and prelysis Weigh 200 mg of stool sample fresh or frozen into a 2 0 ml Safe Lock Tube and add 1 2 ml Lysis Buffer P to each stool sample Vortex vigorously for 1 min Even if you use less starting material perform the protocol like described Important If the sample is liquid pipet 200 ul into the 2 0 ml Safe Lock Tube Cut off the end of the pipet tip to make pipetting easier If the sample is frozen use a scalpel or spatula to scrape bits of stool into the provided 2 0 ml Safe Lock Tube on ice Take care that this samples do not thaw until Lysis Buffer P is added otherwise the DNA in the sample may degrade After addition of the buffer the following steps can be performed at RT or like recommended Incubate the sample for 10 min at RT under continuous shaking at 900 rpm Centrifuge t
46. to 400 mg fresh and frozen human or animal stool samples or from other sample types with high concentrations of PCR inhibiting components The purified DNA can be used for in vitro diagnostic analysis The PSP Spin Stool DNA Plus Kit is an integrated system for collection transportation and storage of fecal samples and subsequent DNA purification The kit has been designed for isolation of DNA from pathogenic microorganisms as well as for isolation of DNA from the host organism Furthermore it is possible to extract nucleic acids from food and feed residues of plant or animal origin from the stool sample The protocols for the isolation and all buffers are optimized for a high yield as well as a high purity All hands on steps are reduced to a minimum Any diagnostic results generated using the sample preparation procedure in conjunction with any downstream diagnostic assays should be interpreted with regard to other clinical or laboratory finding THE PRODUCT IS INTENDED FOR USE BY PROFESSIONALS ONLY SUCH AS TECHNICIANS PHYSICIANS AND BIOLOGISTS TRAINED IN MOLECULAR BIOLOGICAL TECHNIQUES It is designed to be used with any downstream application employing enzymatic amplification or other enzymatic modifications of DNA followed by signal detection or amplification Any diagnostic results generated by using the sample preparation procedure in conjunction with any downstream diagnostic assay should be interpreted with regard to other clinical
47. to extensive quality control procedures according to ISO 9001 2000 and ISO EN 13485 and are warranted to perform as described herein Any problems incidents or defects shall be reported to STRATEC Molecular immediately upon detection thereof The chemicals and the plastic parts are for laboratory use only they must be stored in the laboratory and must not be used for purposes other than intended The Product with its contents is unfit for consumption 6 PSP Spin Stool DNA Kit 0515 PSP Spin Stool DNA Plus Kit 0515 Safety information When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles and avoid skin contact Heed the legal requirements for working with biological material For more information please consult the appropriate material safety data sheets MSDS These are available online in convenient and compact PDF format at www stratec com for each STRATEC Molecular kit and whose kit component If the buffer bottles are damaged or leaking wear gloves and protective goggles when discarding the bottles in order to avoid any injuries STRATEC Molecular has not tested the liquid waste generated by the PSP Spin Stool DNA Kit and the PSP Spin Stool DNA Plus Kit procedures for residual infectious materials Contamination of the liquid waste with residual infectious materials is highly unlikely but cannot be excluded completely Therefore liquid waste must be considered infectious and m
48. ume 160 ml Wash Buffer Il 15 ml ready to use 18 ml final volume 60 ml 2x 45 ml final volume 2 x 150 ml Elution Buffer 2 ml 15 ml 60 ml 2 0 ml Safe Lock Tubes 10 2x50 10 x 50 RTA Spin Filter Set 5 50 5 x 50 RTA Receiver Tubes 2x5 2x50 10 x 50 1 5 ml Receiver Tubes 2x5 2x50 10 x 50 Manual i 1 1 Initial steps Add 250 ul ddH20 to Add 21 ml 99 7 Isopropanol Add 84 ml 99 7 Isopropanol Proteinase K mix thoroughly until completely dissolving and store the tube at 20 C Incubate the needed amount of Elution Buffer at 70 C ina thermomixer to the Binding Buffer A Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times Add 1 5 ml ddH20 to Proteinase K mix thoroughly until completely dissolving and store the tube at 20 C Add 30 ml of 96 100 ethanol to the bottle Wash Buffer I and always keep the bottle firmly closed Add 42 ml of 96 100 ethanol to the bottle Wash Buffer Il mix thoroughly and always keep the bottle firmly closed Preheat needed amount of Elution Buffer at 70 C ina thermomixer to the Binding Buffer A Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times Add 1 5 ml ddH20 to each tube Proteinase K mix thoroughly until completely dissolving and store the tube at 20 C Add 80 ml of 96 100 ethanol to the bottle Wash Buffer I and
49. ust be handled and discarded according to local safety regulation Below is listed European Community risk and safety phrases for the components of the PSP Spin Stool DNA Kit and of the PSP Spin Stool DNA Plus Kit to which they apply Lysis Buffer P Wash Buffer danger warning H319 P305 351 338 H302 312 332 412 EUH032 P273 Proteinase K OD danger H315 319 334 335 P280 305 351 338 310 405 H319 Causes serious eye irritation H315 Causes skin irritation H334 May cause allergy or asthma symptoms or breathing difficulties if inhaled H335 May cause respiratory irritation H302 Harmful if swallowed H312 Harmful in contact with skin H332 Harmful if inhaled H412 Harmful to aquatic life with long lasting effects EUH032 Contact with acids liberates very toxic gas P233 Keep container tightly closed P305 P351 P338 IF IN EYES Rinse cautiously with water for several minutes Remove contact lenses if present and easy to do Continue rinsing P280 Wear protective gloves protective clothing eye protection face protection P310 Immediately call a POISON CENTER or doctor physician P405 Store locked up P273 Avoid release to the environment Emergency medical information can be obtained 24 hours a day from infotrac outside of USA 1 352 323 3500 in USA 1 800 535 5053 7 PSP Spin Stool DNA Kit 0515 PSP Spin Stool DNA Plus Kit 0515 Product characteristic of the PSP Spin Sto
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