User manual PSP Spin Stool DNA Kit/ PSP Spin Stool DNA Plus Kit/ PSP Spin Stool DNA Basic Kit

Transcription

1 User manual PSP Spin Stl DNA Kit/ PSP Spin Stl DNA Plus Kit/ PSP Spin Stl DNA Basic Kit fr (cllectin, strage, stabilizatin) and purificatin f ttal DNA frm fresh r frzen stl samples (including Stl Cllectin Tubes with Stl DNA Stabilizer) x00 IVD REF x x00 STRATEC Mlecular GmbH, D Berlin

2 Instructin fr the PSP Spin Stl DNA Kit The PSP Spin Stl DNA Kit prvides fast and easy purificatin f ttal DNA frm max. 200 mg f fresh r frzen stl samples using the Invisrb technlgy. The purified DNA is f high quality and well suited fr use in in-vitr diagnstic analysis. Instructin fr PSP Spin Stl DNA Plus Kit The PSP Spin Stl DNA Plus Kit is an integrated system fr cllectin, transprtatin and strage f stl samples and subsequent DNA purificatin. The kit has been designed fr islatin f DNA frm pathgenic micrrganisms, as well as fr islatin f DNA frm the hst rganism. Furthermre, it is pssible t extract nucleic acids frm fd and feed residues f plant r animal rigin frm the stl sample. The purified DNA is ideal fr reliable use in PCR and ther dwnstream enzymatic reactins. Instructin fr PSP Spin Stl DNA Basic Kit The PSP Spin Stl DNA Basic Kit is a DNA purificatin system frm stl samples in cmbinatin with the crrespnding Stl Cllectin Tubes with DNA Stabilizer (extra mdule). The kit has been designed fr islatin f DNA frm pathgenic micrrganisms, as well as fr islatin f DNA frm the hst rganism. Furthermre, it is pssible t extract nucleic acids frm fd and feed residues f plant r animal rigin frm the stl sample. The purified DNA is ideal fr reliable use in PCR and ther dwnstream enzymatic reactins. The kits are neither validated fr the islatin f genmic DNA frm cultured r islated cells, frm tissues, swabs, dried bld stains, r cell free bdy fluids, like synvial fluid and urine r the purificatin f RNA. The applicatin f the kits fr islatin and purificatin f viral DNA has nt been evaluated. IVD Cmpliance with EU Directive 98/79/EC n in vitr medical devices. Nt fr in-vitr diagnstic use in cuntries where the EU Directive 98/79/EC n in vitr medical devices is nt recgnized. Trademarks: Invisrb, PSP, InviMag, Eppendrf. Registered marks, trademarks, etc. used in this dcument, even when nt specifically marked as such, are nt t be cnsidered unprtected by law. The Invisrb technlgy is cvered by patents and patent applicatins: US 6,110363, US 6,043,354, US 6,037,465, EP , WO , WO , EP , DE , DE , WO Invisrb, PSP and InviMag are registered trademarks f STRATEC Bimedical AG. 2018, STRATEC Mlecular, all rights reserved. 1 PSP Spin Stl System 0218

3 Cntents Kit cmpnents f PSP Spin Stl DNA Kit 3 Kit cmpnents f PSP Spin Stl DNA Plus Kit 4 Kit cmpnents f PSP Spin Stl DNA Basic Kit 5 Stl Cllectin Sets 6 Symbls 6 Strage 6 Quality cntrl 7 Intended use 7 Prduct use limitatins 8 Safety infrmatin 8 Prduct characteristic f PSP Spin Stl DNA Kit 10 Prduct characteristic f PSP Spin Stl DNA Plus Kit 11 Prduct characteristic f PSP Spin Stl DNA Basic Kit 11 Principle and prcedure 12 Sampling and strage 12 Prcedure 13 Yield and quality f ttal DNA 13 Imprtant ntes 13 Imprtant pints befre starting a prtcl 13 Preparing reagents and buffers 14 Equipment and reagents t be supplied by user 14 Imprtant indicatins 15 Scheme f PSP Spin Stl DNA Kit 16 Prtcl 1: Islatin f ttal DNA frm up t 200 mg stl samples 17 with and withut enrichment f bacterial DNA Prtcl 2: Islatin f ttal DNA frm up t 200 mg stl samples 19 frm difficult t lyse bacteria Scheme f PSP Spin Stl DNA Plus Kit & PSP Spin Stl DNA Basic Kit 21 Prtcl 1: Cllectin f the stl sample and stabilizatin 22 Prtcl 2: Islatin f ttal DNA frm 1.4 ml stabilized stl 22 hmgenate with and withut enrichment f bacterial DNA Prtcl 3: Islatin f ttal DNA frm up t 200 mg stl samples 24 frm difficult t lyse bacteria Trubleshting 26 Appendix 29 General ntes n handling DNA 29 Determinatin f cncentratin, yield and purity f DNA 29 Ordering infrmatin 30 Supplemental nt validated prtcls, nt fr diagnstic use 31 Supplemental prtcl fr Pst purificatin f DNA cntaining inhibitrs 31 2 PSP Spin Stl System 0218

4 Kit cmpnents f PSP Spin Stl DNA Kit 5 extractins 50 extractins 250 extractins Catalg N Lysis Buffer P 3 x 2 ml 120 ml 2 x 160 ml Zircnia Beads II 1 vial 2 vial 8 vials InviAdsrb x 50 Prteinase K fr 250 µl wrking slutin fr 1.5 ml wrking slutin fr 5 x 1.5 ml wrking slutin Binding Buffer A 2 x 1 ml (ready t use) 9 ml (final vlume 30 ml) 36 ml (final vlume 120 ml) Wash Buffer I 15 ml (ready t use) 30 ml (final vlume 60 ml) 80 ml (final vlume 160 ml) Wash Buffer II 15 ml (ready t use) 18 ml (final vlume 60 ml) 2 x 45 ml (final vlume 2 x 150 ml) Elutin Buffer 2 ml 15 ml 60 ml 2.0 ml Safe-Lck-Tubes 10 2 x 50 2 x 250 RTA Spin Filter Set x 50 RTA Receiver Tubes 2 x 5 2 x x ml Receiver Tubes 2 x 5 2 x x 50 Manual Initial steps Add 250 µl ddh2o t Prteinase K, mix thrughly until cmpletely disslving Incubate the needed amunt f Elutin Buffer at 70 C in a thermmixer Add 21 ml 99.7% Isprpanl t the Binding Buffer A. Mix by intensive shaking by inverting fr 1 min. Shrtly befre use mix by inverting several times. Add 1.5 ml ddh2o t Prteinase K, mix thrughly until cmpletely disslving Add 30 ml f % ethanl t the bttle Wash Buffer I mix thrughly and always keep the bttle firmly clsed Add 42 ml f % ethanl t the bttle Wash Buffer II, mix thrughly and always keep the bttle firmly clsed Incubate the needed amunt f Elutin Buffer at 70 C in a thermmixer Add 84 ml 99.7% Isprpanl t each Binding Buffer A. Mix by intensive shaking by inverting fr 1 min. Shrtly befre use mix by inverting several times. Add 1.5 ml ddh2o t each tube Prteinase K, mix thrughly until cmpletely disslving Add 80 ml f % ethanl t the bttle Wash Buffer I and always keep the bttle firmly clsed Add 105 ml f % ethanl t each bttle Wash Buffer II, mix thrughly and always keep the bttle firmly clsed Incubate the needed amunt f Elutin Buffer at 70 C in a thermmixer 3 PSP Spin Stl System 0218

5 Kit cmpnents f PSP Spin Stl DNA Plus Kit 5 extractins 50 extractins 250 extractins Catalg N Stl Cllectin Tubes with DNA Stabilizer 5 2 x x 25 InviAdsrb x 50 Zircnia Beads II 1 vial 2 vial 8 vials Prteinase K fr 250 µl wrking slutin fr 1.5 ml wrking slutin fr 5 x 1.5 ml wrking slutin Binding Buffer A 3 x 1 ml (ready t use) 9 ml (final vlume 30 ml) 36 ml (final vlume 120 ml) Wash Buffer I 15 ml (ready t use) 30 ml (final vlume 60 ml) 80 ml (final vlume 160 ml) Wash Buffer II 15 ml (ready t use) 18 ml (final vlume 60 ml) 2 x 45 ml (final vlume 2 x 150 ml) Elutin Buffer 2 ml 15 ml 60 ml 2.0 ml Safe-Lck-Tubes 10 2 x 50 2 x 250 RTA Spin Filter Set x 50 RTA Receiver Tubes 2 x 5 2 x x ml Receiver Tubes 2 x 5 2 x x 50 Manual Initial steps Add 250 µl ddh2o t Prteinase K, mix thrughly until cmpletely disslving Incubate the needed amunt f Elutin Buffer at 70 C in a thermmixer Add 21 ml 99.7% Isprpanl t the Binding Buffer A. Mix by intensive shaking by inverting fr 1 min. Shrtly befre use mix by inverting several times. Add 1.5 ml ddh2o t Prteinase K, mix thrughly until cmpletely disslving Add 30 ml f % ethanl t the bttle Wash Buffer I and always keep the bttle firmly clsed! Add 42 ml f % ethanl t the bttle Wash Buffer II, mix thrughly and always keep the bttle firmly clsed! Preheat needed amunt f Elutin Buffer at 70 C in a thermmixer Add 84 ml 99.7% Isprpanl t the Binding Buffer A. Mix by intensive shaking by inverting fr 1 min. Shrtly befre use mix by inverting several times. Add 1.5 ml ddh2o t each tube Prteinase K, mix thrughly until cmpletely disslving Add 80 ml f % ethanl t the bttle Wash Buffer I and always keep the bttle firmly clsed! Add 105 ml f % ethanl t each bttle Wash Buffer II, mix thrughly and always keep the bttle firmly clsed! Preheat needed amunt f Elutin Buffer at 70 C in a thermmixer 4 PSP Spin Stl System 0218

6 Kit cmpnents f PSP Spin Stl DNA Basic Kit 50 extractins 250 extractins Catalg N InviAdsrb 50 5 x 50 Zircnia Beads II 2 vial 8 vials Prteinase K fr 1.5 ml wrking slutin fr 5 x 1.5 ml wrking slutin Binding Buffer A 9 ml (final vlume 30 ml) 36 ml (final vlume 120 ml) Wash Buffer I 30 ml (final vlume 60 ml) 80 ml (final vlume 160 ml) Wash Buffer II 18 ml (final vlume 60 ml) 2 x 45 ml (final vlume 2 x 150 ml) Elutin Buffer 15 ml 60 ml 2.0 ml Safe-Lck-Tubes 2 x 50 2 x 250 RTA Spin Filter Set 50 5 x 50 RTA Receiver Tubes 2 x x ml Receiver Tubes 2 x x 50 Manual 1 1 Initial steps Add 21 ml 99.7% Isprpanl t the Binding Buffer A. Mix by intensive shaking by inverting fr 1 min. Shrtly befre use mix by inverting several times. Add 1.5 ml ddh2o t Prteinase K, mix thrughly until cmpletely disslving Add 30 ml f % ethanl t the bttle Wash Buffer I and always keep the bttle firmly clsed! Add 42 ml f % ethanl t the bttle Wash Buffer II, mix thrughly and always keep the bttle firmly clsed! Preheat needed amunt f Elutin Buffer at 70 C in a thermmixer Add 84 ml 99.7% Isprpanl t the Binding Buffer A. Mix by intensive shaking by inverting fr 1 min. Shrtly befre use mix by inverting several times. Add 1.5 ml ddh2o t each tube Prteinase K, mix thrughly until cmpletely disslving Add 80 ml f % ethanl t the bttle Wash Buffer I and always keep the bttle firmly clsed! Add 105 ml f % ethanl t each bttle Wash Buffer II, mix thrughly and always keep the bttle firmly clsed! Preheat needed amunt f Elutin Buffer at 70 C in a thermmixer Imprtant: Fr stl sample cllectin, stabilizatin and transprt the STRATEC Mlecular Stl Cllectin Tubes with DNA Stabilizer r the Stl Stabilizer reagent shuld be rdered additinally. We recmmend rdering separately the PSP Spin Stl DNA Basic Kit and Stl Cllectin Tubes with DNA Stabilizer if samples are cllected at different places r perids (see Ordering infrmatin at page 30). 5 PSP Spin Stl System 0218

7 Stl Cllectin Sets Stl DNA Stabilizer ml Stl Cllectin Tube with DNA Stabilizer tubes Stl Cllectin Tube with DNA Stabilizer tubes Symbls Manufacturer Lt number Catalgue number Attentin: D nt cmbine cmpnents f different kits, unless the lt numbers are identical! Expiry date Cnsult perating instructins Temperature limitatin D nt reuse Humidity limitatin Strage All buffers and kit cntents f the PSP Spin Stl DNA Kit, the PSP Spin Stl DNA Plus Kit and the PSP Spin Stl DNA Basic Kit, except disslved Prteinase K shuld be stred at rm temperature and are stable fr at least 12 mnths. Rm temperature (RT) is defined as range frm C. Befre every use make sure that all cmpnents have rm temperature. If there are any precipitates within the prvided slutins slve these precipitates by warming carefully (up t 30 C). Stl Cllectin Tube with DNA Stabilizer: If there are any precipitates within the prvided slutins slve these precipitates by warming carefully. Please incubate fr 5 min. in a 30 C water bath r in a 37 C incubatr. After cmplete disslving mix the buffer by gently shaking. The functinality f the buffer is nt influenced by the precipitates. Prteinase K: Disslved Prteinase K must be stred at 2-8 C fr up t tw mnths. Fr lnger strage 20 C is recmmended, freeze-thaw nce nly. S the disslved Prteinase K is stable as indicated n the kit package. Wash Buffers charged with ethanl shuld be apprpriately sealed and stred at rm temperature. Binding Buffer charged with isprpanl shuld be apprpriately sealed and stred at rm temperature. 6 PSP Spin Stl System 0218

8 Quality cntrl and prduct warranty STRATEC Mlecular warrants the crrect functin f the PSP Spin Stl DNA Kit, the PSP Spin Stl DNA Plus Kit and the PSP Spin Stl DNA Basic Kit fr applicatins as described in this manual. Purchaser must determine the suitability f the Prduct fr its particular use. Shuld any Prduct fail t perfrm the applicatins as described in the manual, STRATEC Mlecular will check the lt and if STRATEC Mlecular investigates a prblem in the lt, STRATEC Mlecular will replace the Prduct free f charge. STRATEC Mlecular reserves the right t change, alter, r mdify any Prduct t enhance its perfrmance and design at any time. In accrdance with STRATEC Mlecular s EN ISO 9001 and EN ISO certified Quality Management System the perfrmance f all cmpnents f the PSP Spin Stl DNA Kit, the PSP Spin Stl DNA Plus Kit and the PSP Spin Stl DNA Basic Kit have been tested separately against predetermined specificatins rutinely n lt-t-lt t ensure cnsistent prduct quality. If yu have any questins r prblems regarding any aspects f PSP Spin Stl DNA Kit, the PSP Spin Stl DNA Plus Kit and the PSP Spin Stl DNA Basic Kit r ther STRATEC Mlecular prducts, please d nt hesitate t cntact us. A cpy f STRATEC Mlecular s terms and cnditins can be btained upn request r are presented at the STRATEC Mlecular webpage. Fr technical supprt r further infrmatin please cntact: frm Germany +49-(0) / 2910 frm abrad +49-(0) r cntact yur lcal distributr. Intended use The PSP Spin Stl DNA Kit has been designed fr fast and efficient purificatin f genmic and micrbial DNA frm up t 200 mg fresh and frzen human r animal stl samples r frm ther sample types with high cncentratins f PCR inhibiting cmpnents. The purified DNA can be used fr in-vitr diagnstic analysis. The PSP Spin Stl DNA Plus Kit is an integrated system fr cllectin, transprtatin and strage f fecal samples and subsequent DNA purificatin. The kit has been designed fr islatin f DNA frm pathgenic micrrganisms, as well as fr islatin f DNA frm the hst rganism. Furthermre, it is pssible t extract nucleic acids frm fd and feed residues f plant r animal rigin frm the stl sample. If samples are cllected at different places r perid we recmmend rdering separately the PSP Spin Stl DNA Basic Kit and Stl Cllectin Tube with DNA Stabilizer. The prtcls fr the islatin and all buffers are ptimized fr a high yield as well as a high purity. All hands n steps are reduced t a minimum. Any diagnstic results generated using the sample preparatin prcedure in cnjunctin with any dwnstream diagnstic assays shuld be interpreted with regard t ther clinical r labratry finding. THE PRODUCT IS INTENDED FOR USE BY PROFESSIONALS ONLY, SUCH AS TECHNICIANS, PHYSICIANS AND BIOLOGISTS TRAINED IN MOLECULAR BIOLOGICAL TECHNIQUES. It is designed t be used with any dwnstream applicatin emplying enzymatic amplificatin r ther enzymatic mdificatins f DNA fllwed by signal detectin r amplificatin. Any diagnstic results generated by using the sample preparatin prcedure in cnjunctin with any dwnstream diagnstic assay shuld be interpreted with regard t ther clinical r labratry findings. T minimize irregularities in diagnstic results, adequate cntrls fr dwnstream applicatins shuld be used. The kit is in cmpliance with EU Directive 98/79/EC n in vitr medical devices. But it is nt fr in-vitr diagnstic use in cuntries where the EU Directive 98/79/EC n in vitr medical devices is nt recgnized. 7 PSP Spin Stl System 0218

9 Prduct use limitatin The kit is nt validated fr the islatin f genmic DNA frm cultured r islated cells, frm tissue, swabs, dried bld stains, r cell free bdy fluid, like synvial fluid and urine, r the purificatin f RNA. The applicatin f the kit fr islatin and purificatin f viral DNA has nt been evaluated. The included chemicals are nly useable nce. Differing f starting material r flw trace may lead t inperability; therefre neither a warranty nr guarantee in this case will be given, neither implied nr express. The user is respnsible t validate the perfrmance f the STRATEC Mlecular Prduct fr any particular use. STRATEC Mlecular des nt prvide fr validatin f perfrmance characteristics f the Prduct with respect t specific applicatins. STRATEC Mlecular prducts may be used e.g. in clinical diagnstic labratry systems under fllwing cnditins: If used in the US, based n the cnditin that the cmplete diagnstic system f the labratry has been validated pursuant t CLIA 88 regulatins. Fr ther cuntries based n the cnditin that the labratry has been validated pursuant t equivalents accrding t the respective legal basis. All Prducts sld by STRATEC Mlecular are subject t extensive quality cntrl prcedures (accrding t EN ISO 9001 and EN ISO 13485) and are warranted t perfrm as described herein. Any prblems, incidents r defects shall be reprted t STRATEC Mlecular immediately upn detectin theref. The chemicals and the plastic parts are fr labratry use nly; they must be stred in the labratry and must nt be used fr purpses ther than intended. The Prduct with its cntents is unfit fr cnsumptin. Safety infrmatin When wrking with chemicals, always wear a suitable lab cat, dispsable glves and prtective gggles and avid skin cntact. Heed the legal requirements fr wrking with bilgical material! Fr mre infrmatin, please cnsult the apprpriate material safety data sheets (MSDS). These are available nline in cnvenient and cmpact PDF frmat at fr each STRATEC Mlecular kit and whse kit cmpnent. If the buffer bttles are damaged r leaking, wear glves and prtective gggles when discarding the bttles in rder t avid any injuries. STRATEC Mlecular has nt tested the liquid waste generated by the PSP Spin Stl DNA Kit and the PSP Spin Stl DNA Plus Kit prcedures fr residual infectius materials. Cntaminatin f the liquid waste with residual infectius materials is highly unlikely, but cannt be excluded cmpletely. Therefre, liquid waste must be cnsidered infectius and must be handled and discarded accrding t lcal safety regulatin. 8 PSP Spin Stl System 0218

10 Belw is listed Eurpean Cmmunity risk and safety phrases fr the cmpnents f the PSP Spin Stl DNA Kit, PSP Spin Stl DNA Plus Kit and PSP Spin Stl DNA Basic Kit t which they apply Stl DNA Stabilizer Binding Buffer A (ready t use) Warning H319 H412.-P280- P P273 Lysis Buffer P Danger H225-H319-H336-P210-P233-P305+P351+P338 Prteinase K Warning H319 H412.-P280- P P273 Wash Buffer I (ready t use) Danger H P280-P305-P351-P338 Wash Buffer I Danger H225-P403+P233 Warning H302-H412-P280-P305-P351-P338-P273-EUH032 Wash Buffer II (ready t use) Danger H225-P403+P233 H225: Highly flammable liquid and vapur. H302: Harmful if swallwed. H315: Causes skin irritatin.h319: Causes serius eye irritatin. H319: Causes serius eye irritatin. H334: May cause allergy r asthma symptms r breathing difficulties if inhaled. H335: May cause respiratry irritatin. H336: May cause drwsiness r dizziness. H412: Harmful t aquatic life with lng lasting effects. P210: Keep away frm heat, ht surfaces, sparks, pen flames and ther ignitin surces. N smking. P233: Keep cntainer tightly clsed. P273: Avid release t the envirnment. P280: Wear prtective glves/prtective clthing/eye prtectin/face prtectin. P305+P351+P338: IF IN EYES: Rinse cautiusly with water fr several minutes. Remve cntact lenses, if present and easy t d. Cntinue rinsing. P403+P233: Stre in a well-ventilated place. Keep cntainer tightly clsed. EUH032: Cntact with acids liberates very txic gas. Emergency medical infrmatin can be btained 24 hurs a day frm inftrac: utside f USA: in USA : PSP Spin Stl System 0218

11 Prduct characteristic f the PSP Spin Stl DNA Kit Starting material Yield Time Rati max. 200 mg fecal sample up t 50 µg (depends n starting material) abut 45 min (incl. lysis time) A 260 : A 280 1,4 1,8 The PSP Spin Stl DNA Kit allws rapid and efficient islatin f high quality DNA frm up t 200 mg f fresh r frzen stl sample. Stl samples typically cntain many cmpunds that can degrade DNA and inhibit dwnstream enzymatic reactins. T ensure the remval f these cntaminants the PSP Spin Stl DNA Kit cntains tubes with InviAdsrb and ptimized essential washing cnditins t remve all ptent inhibitrs very efficiently. S the simple PSP Spin prcedure yields pure DNA ready t use in less than 1h. A rigrus prelysis step using Zircnia Beads II with ptimized prelysis buffer under high temperatures is fllwed by a preincubatin f the sample with InviAdsrb t remve PCR inhibitrs. Undisslved particles and PCR inhibitrs bund t InviAdsrb are remved by a centrifugatin step. The fllwing Prteinase K digestin ensures high yields als frm gram psitive bacteria. Stl cntains a range f DNA e.g. hst DNA frm cln epithelial cells, parasite DNA, bacterial DNA, DNA frm fd r DNA frm gastrintestinal pathgens. The chise f different lysis cnditins allws the enrichment r a reductin f the cntent f bacterial DNA in the ttal DNA. All impurities are remved very efficiently during washing steps and the purified DNA is eluted directly in a lw-salt buffer. N phenl/chlrfrm extractin r ethanl precipitatin is necessary. The kit prvides reprducible recvery rates f highly purified DNA, ready t use in any dwnstream applicatin. The islated DNA can be stred at -20 C fr later use. Due t the high purity, the islated ttal DNA is suitable fr a brad panel f dwnstream applicatins (see belw) r can be stred at -80 C fr subsequent use. PCR applicatins RFLP-analysis Hybridizatin Genetic typing Pathgen typing Mutatin analysis N txic r hazardus chemicals like phenl/chlrfrm r ß-Mercaptethanl are used. Traditinal time-killing prcedures can be replaced using the PSP Spin Stl DNA Kit. T increase rbustness f PCR assays using DNA islated frm stl samples, the additin f BSA t a final cncentratin f 0.1 µg/ µl t the PCR mixtures is recmmended. In DNA eluates frm feces, the rati f target DNA t backgrund DNA is ften very lw. 10 PSP Spin Stl System 0218

12 Prduct characteristic f the PSP Spin Stl DNA Plus Kit Starting material Yield Time Rati 1.4 ml Stl DNA Stabilizer with stl hmgenate up t 50 µg (depends n starting material) abut 45 min (incl. lysis time) A 260 : A 280 1,4 1,8 The PSP Spin Stl DNA Plus Kit cmbines the cllectin f stl samples, the strage and stabilizatin f the stl specimen withut any degradatin f the DNA with a very efficient and fast islatin f high quality ttal DNA. The PSP Spin Stl DNA Plus Kit can be used fr islatin f DNA frm pathgenic micrrganism, as well as fr islatin f DNA frm hst rganism. The PSP Spin Stl DNA Plus Kit uses a stabilizing reagent, the Stl DNA Stabilizer, which enables the strage f the stl samples after cllectin withut cling under ambient temperature fr at least 3 mnth. The system cmbines the use f the Stl Cllectin Tubes prefilled with the DNA Stabilizer fr cllectin, strage and stabilizatin f the stl specimen withut any degradatin f the DNA during transprtatin and the prelysis f bacteria with a very efficient and fast islatin f high quality DNA frm stl sample. In additin t the inactivatin f DNases, the Stl DNA Stabilizer preserves the micrrganism titer. Furthermre, the Stl DNA Stabilizer enables prelysis f gram psitive r negative bacteria. Fr the DNA extractin prcess nly a small amunt f the ttal vlume will be used, the residual sample can be used fr further extractins r a lng term strage at -20 C. Stl samples typically cntain many cmpnents that can degrade DNA and inhibit dwn stream enzymatic reactins. Therefre the PSP Spin Stl DNA Plus Kit cntains InviAdsrb, a reagent that efficiently adsrbs these cmpnents at the beginning f the purificatin prcess and additinally ptimized essential washing cnditins fr the final remval f the last traces f all ptent inhibitrs. (See mre t the prcedure at page 11 and19.) Due t the high purity, the islated DNA is suitable fr a brad panel f dwnstream applicatins (see belw) r can be stred at 20 C fr later use. PCR applicatins RFLP analysis Hybridizatin Genetic typing Pathgen typing Mutatin analysis Paternity testing T purify high mlecular weight ttal DNA using magnetic beads, STRATEC Mlecular ffers different kits in 15 well r in 96 frmat, the InviMag Stl DNA Mini Kit KFmL and KF96 fr use n the KingFisher wrkstatins. Prduct characteristic f the PSP Spin Stl DNA Basic Kit The PSP Spin Stl DNA Basic Kit is part f the PSP Spin Stl DNA Plus Kit withut cllectin mdule. In cmbinatin with the Stl Cllectin Tube with DNA Stabilizer the PSP Spin Stl DNA Basic Kit has the same prduct characteristics like the PSP Spin Stl DNA Plus Kit. 11 PSP Spin Stl System 0218

13 Principle and prcedure The PSP Spin Stl DNA Kit prcedure cmprises fllwing steps: lysis f sample remval f PCR inhibitrs prtein digestin binding the nucleic acids t the membrane f a spin clumn washing f the spin clumn and hereby eliminatin f cntaminants and ethanl elutin f the nucleic acids After hmgenizatin f the sample in the Lysis Buffer P which inactivates DNases, the human cells and the bacterial cell wall will be lysed differently (depending n the temperature prfile). The lysate will be mixed with InviAdsrb and mst f the PCR inhibiting cmpnents will be remved fllwed by a prtein digestin. After lysis the DNA binds t the membrane, cntaminatins and enzyme inhibitrs are efficiently remved during the fllwing three washing steps and highly purified DNA is eluted in Elutin Buffer r water. The PSP Spin Stl DNA Plus Kit prcedure and the PSP Spin Stl DNA Basic Kit (in cmbinatin with f the Stl Cllectin Tube with DNA Stabilizer) prcedure cmprises sme additinal steps likes: sample cllectin DNA stabilizatin in the sample fllwed by all steps frm the PSP Spin Stl DNA Kit prcedure After sample cllectin and hmgenizatin f the sample in the Stl Cllectin Tube cntaining the DNA stabilizing Lysis Buffer which inactivates DNases, the human and bacterial cells will be lysed differently (depending frm the temperature prfile). The DNA is stable fr mre than 3 mnth and can be transprted t the lab withut degradatin, prelysing bacteria and stpping further bacterial grw. The lysate will be mixed later with InviAdsrb and the mst PCR inhibiting cmpnents will be remved, fllwed by a prtein digestin. After lysis the DNA binds t the membrane, cntaminatins and enzyme inhibitrs are efficiently remved during the fllwing three wash steps and highly purified DNA is eluted in Elutin Buffer r water. Sampling and strage f starting material PSP Spin Stl DNA Kit The cllected fresh stl sample can be stred at ambient temperature fr at least 1-2 hurs at RT, but the high cntent f DNases realize quickly a DNA digestin and degradatin. The sample shuld be quickly added t the Lysis Buffer P r can be stred frzen at 20 C fr weeks. PSP Spin Stl DNA Plus Kit The strage f fresh samples in DNA Stabilizer prvided in the Stl Cllectin Tubes, allw strage at RT fr abut 3 mnth. The strage f fresh samples in DNA Stabilizer will lead t less degraded DNA and a better yield f bacterial pathgens with difficult t lyse cell walls. Strage time belw 3 mnth has n influence n the quality r the amunt f hst cell DNA. The cllected sample in Stl DNA Stabilizer can als be used immediately fr the islatin f DNA after cllectin. The cllected sample can be refrigerated at 20 C immediately after cllectin r after strage at ambient temperature fr a later use (fr example fr a secnd DNA islatin). STRATEC Mlecular will be released f its respnsibilities if ther sample materials than described in the Intended Use are prcessed r if the sample preparatin prtcls are changed r mdified. 12 PSP Spin Stl System 0218

14 Prcedure Lysis Stl samples are lysed in Lysis Buffer P under denaturing cnditins at high temperatures. Human cells lyse efficiently at RT, bacterial cells and thse f ther pathgens in the stl sample are efficiently lysed by incubatin at 95 C. This is recmmended fr detectin f cells that are difficult t lyse (e.g. gram psitive bacteria). Nte: The ttal DNA cncentratin in the lysate will be increased 3-5 fld by lysis at 95 C and the rati f nn human t human DNA will increase. Remval f PCR inhibitrs After lysis prcedure DNA damaging substances and PCR inhibitrs which are present in the feces are adsrbed efficiently t the InviAdsrb matrix. InviAdsrb is prvided very cnvenient in safe lck tubes and the lysate must nly be mixed with the matrix. The bund cntaminatins and cell debris are pelleted by centrifugatin. The supernatant cntains the precleaned DNA. Prtein digestin Prteinase K is added t the supernatant t digest and degrade prteins during the incubatin at 70 C. Binding f ttal DNA After adding Binding Buffer A t the supernatant, the mixture is transferred t the spin clumns and nucleic acids are bund t the membrane f the RTA Spin Filter during a brief centrifugatin step. Optimal salt cncentratins and ph cnditins in the lysate ensure that remains f digested prteins and ther cntaminatins, which can inhibit dwnstream enzymatic reactins, are nt retained n the Invisrb membrane. Remving residual cntaminants DNA bund t the Invisrb membrane is washed in three centrifugatin steps. Cntaminants are efficiently and cmpletely remved using Wash Buffer I and II, while the nucleic acids remain bund t the membrane. Elutin The nucleic acids are eluted in lw salt buffer frm the membrane using µl Elutin Buffer. The eluted nucleic acids are ready t use in different subsequent tests. Yield and quality f ttal DNA The amunt f purified DNA in the PSP Spin Stl DNA Kit prcedure frm feces depends n the health status f the dnr, bacteria cntent, sample surce, transprt, strage, and age. A typical yield is µg, a typical DNA cncentratin is ng/ µl. Yield and quality f islated genmic DNA is suitable fr any mlecular-diagnstic detectin system. The diagnstic tests shuld be perfrmed accrding t manufacturers specificatins. Imprtant ntes Imprtant pints befre starting a prtcl Immediately upn receipt f the Prduct, inspect the Prduct and its cmpnents as well as the package fr any apparent damages, crrect quantities and quality. If there are any uncnfrmities yu have t ntify STRATEC Mlecular in writing with immediate effect upn inspectin theref. If buffer bttles are damaged, cntact the STRATEC Mlecular Technical Services r yur lcal distributr. In case f liquid spillage, refer t Safety Infrmatin (see page 7). D nt use damaged kit cmpnents, since their use may lead t pr kit perfrmance. 13 PSP Spin Stl System 0218

15 Always change pipet tips between liquid transfers. T avid crss-cntaminatin, we recmmend the use f aersl-barrier pipet tips. All centrifugatin steps are carried ut at rm temperature. When wrking with chemicals, always wear a suitable lab cat, dispsable glves, and prtective gggles. Discard cntaminated glves. D nt cmbine cmpnents f different kits unless the lt numbers are identical. Avid micrbial cntaminatin f the kit reagents. T minimize the risk f infectins frm ptentially infectius material, we recmmend wrking under laminar air-flw until the samples are lysed. This kit shuld nly be used by trained persnnel. Preparing reagents and buffers fr the PSP Spin Stl System 1. Adjust the thermmixer t 70 C. 2. Disslve Prteinase K in ddh 2 O. 3. Warm up the needed amunt f Elutin Buffer t 70 C, ( µl Elutin Buffer are needed per sample). 4. Heat heating blcks (e.g. thermmixer) t 70 C and 95 C. 5. Label the needed amunt f 2.0 ml RTA Spin Filter Sets. 6. Label the needed amunt f 1.5 ml Receiver Tubes (per sample: 1 Receiver Tube), add the needed amunt f ethanl t the Wash Buffer I and II (see Kit cntents, page 3/4/5). 5 ttal DNA extractins: add 250 µl ddh2o t Prteinase K, mix thrughly until cmpletely disslving Binding Buffer A, Wash Buffer I and II are ready t use 50 ttal DNA extractins: add 21 ml 99.7% Isprpanl t the Binding Buffer A. Mix by intensive shaking by inverting fr 1 min. Shrtly befre use mix by inverting several times. add 1.5 ml ddh2o t Prteinase K, mix thrughly until cmpletely disslving add 30 ml % ethanl t the bttle Wash Buffer I add 42 ml % ethanl t each bttle Wash Buffer II mix thrughly and always keep the bttle firmly clsed 250 ttal DNA-extractins: add 84 ml 99.7% Isprpanl t the Binding Buffer A. Mix by intensive shaking by inverting fr 1 min. Shrtly befre use mix by inverting several times. add 1.5 ml ddh2o t Prteinase K, mix thrughly until cmpletely disslving add 80 ml % ethanl t each bttle Wash Buffer I add 105 ml % ethanl t each bttle Wash Buffer II mix thrughly and always keep the bttle firmly clsed Equipment and reagents t be supplied by user When wrking with chemicals, always wear a suitable lab cat, dispsable glves and prtective gggles. Fr mre infrmatin, please cnsult the apprpriate material safety data sheets (MSDS). (See ur webpage: Micrcentrifuge Thermmixer (fr 95 C) Measuring cylinder (250 ml) Dispsable glves Pipet with tips Reagents reservirs fr multichannel pipets % ethanl ddh 2 O Vrtexer r ther hmgenizer Isprpanl 14 PSP Spin Stl System 0218

16 The PSP Spin Stl DNA Kit, the PSP Spin Stl DNA Plus Kit and the PSP Spin Stl DNA Basic Kit are validated with 2-Prpanl; Rtipuran >99.7%, p.a., ACS, ISO (Order n. 6752) frm Carl Rth * * Pssible suppliers fr Isprpanl: Carl Rth 2-Prpanl Rtipuran >99.7%, p.a., ACS, ISO Order n Applichem 2-Prpanl für die Mlekularbilgie Order n. A3928 Sigma 2-Prpanl Order n L-F Imprtant indicatins 1. The kit prcedure is als suitable fr purifying DNA frm very small amunts f starting material. If the sample has less than 5 ng DNA (>1.000 cpies), 3-5 µg Carrier (a hmplymer such as ply da, ply-dt r genmic DNA) shuld be added t the starting material. Ensure that the Carrier DNA des nt interfere with dwnstream applicatin. In rder t prevent any interference f the carrier with the dwnstream applicatin, a RNA carrier can be used. This can be remved by RNase digestin. The carrier shuld be added t the lysis buffer befre preparatin r t the stabilizatin buffer, never add t the stl directly. 2. Invisrb RTA Spin filter can als purify lw amunts f RNA besides DNA. Fr the eliminatin f RNA (if necessary) add 20 µl RNase A (10 mg/ml) befre adding the Binding Buffer A. Vrtex briefly and incubate the sample at rm temperature fr 5 minutes. Then g n as described in the prtcl. Elutin f DNA Fr dwnstream applicatins, that require small starting vlumes, a mre cncentrated eluate may increase assay sensitivity. The elutin can be dne by using a lwer vlume f Elutin Buffer (dwn t 60 µl). This may result in a higher cncentratin f DNA. But lwer vlumes f Elutin Buffer will decrease the yield f DNA. The final vlume f eluate recvered may be up t 5 µl less than the vlume f elutin buffer applied t the spin filter. If lw cncentrated TRIS-buffer affects sensitive dwnstream applicatins, use distilled sterile water fr elutin. Hwever, ensure that the ph f the water is at least 7,0 (deinized water frm certain surces can be acidic). DNA stred in water is subjected t degradatin by acid hydrlysis. Eluting twice with each 100 µl Elutin Buffer is als pssible and gives slightly higher yield f DNA. Handling f the RTA Spin Filter Set Due t the sensitivity f DNA amplificatin technlgies, the fllwing precautins are necessary: t avid crss-cntaminatin between sample preparatin when handling RTA Spin Filter Set carefully apply the sample r slutin t the RTA Spin Filter Set, pipet the sample int the filter withut wetting the rim f the clumn always change pipet tips between liquid transfers, we recmmend the use f aerslbarrier pipet tips avid tuching the RTA Spin Filter membrane with the pipet tip 15 PSP Spin Stl System 0218

17 PSP Spin Stl DNA Kit Scheme f the PSP Spin Stl DNA Kit Please read prtcls prir the start f the preparatin carefully transfer 200 mg f the stl sample int a 2 ml Safe-Lck-Tube, add 1.2 ml Lysis Buffer P, vrtex fr 1 min. fr enrichment f hst DNA: incubate 10 min at RT under shaking fr enrichment f bacterial DNA: incubate 10 min at 95 C n a thermmixer under shaking, add 5 Zircnia Beads II t the hmgenate and vrtex fr 2 min spin dwn at x g ( rpm) fr 1 min transfer the supernatant t the InviAdsrb-Tube mix it by vrtexing fr 15 sec. incubate 1 min at RT spin dwn fr 3 min at full speed transfer the supernatant in a new 1.5 ml Receiver Tube centrifuge the sample again at full speed fr 3 min. Add 25 µl Prteinase K t a 2.0 ml Safe-Lck-Tube transfer 400 µl f the sample supernatant the 2.0 ml Safe-Lck-Tube with Prteinase K mix shrtly by vrtexing incubate fr 10 min at 70 C while cntinuusly shaking n a thermmixer at 900 rpm add 200 µl Binding Buffer A (fllw preparing instructins) t the lysate mix shrtly by vrtexing r pipetting up and dwn transfer the whle mixture t the RTA Spin Filter incubate fr 1 min at RT centrifuge at x g ( rpm) fr 2 min discard the filtrate and the RTA Receiver Tube transfer the RTA Spin Filter in a new RTA Receiver Tube pipet 500 µl Wash Buffer I nt the RTA-Spin Filter centrifuge at x g ( rpm) fr 1 min discard the flw-thrugh and the RTA Receiver Tube put the RTA Spin Filter in a new RTA Receiver Tube pipet 700 µl Wash Buffer II nt the RTA Spin Filter centrifuge at x g ( rpm) fr 1 min. discard the flw-thrugh and reuse the RTA Receiver Tube t eliminate any traces f ethanl, centrifuge again fr 4 min at maximum speed, discard the RTA Receiver Tube transfer the RTA Spin Filter int a new 1.5 ml Receiver Tube pipet µl f Elutin Buffer (preheated t 70 C) directly nt the center f the membrane f the RTA Spin Filter incubate fr 1 min at RT centrifuge at x g ( rpm) fr 1 min discard the RTA-Spin Filter place the eluted ttal DNA immediately n ice and stre at 20 C 16 PSP Spin Stl System 0218

18 PSP Spin Stl DNA Kit Instructins The fllwing ntes are valid fr the fllwing tw prtcls: Nte: The centrifugatin steps were made with the Centrifuge 5415 D frm Eppendrf. The indicated rpm amunts are referring t this centrifuge. Prtcl 1: Islatin f ttal DNA frm up t 200 mg stl samples with and withut enrichment f bacterial DNA Please read prtcls prir the start f the preparatin and cmplete preparing steps!! Attentin: Please be aware, that yu have t prepare the Binding Buffer A see instructin page: 13 Imprtant Nte: 1. Sample hmgenizatin and prelysis Please nte that the majrity f extracted DNA frm stl samples is f bacterial rigin! Heat heating blcks (e.g. thermmixer) t 70 C and 95 C Preheat the Elutin Buffer t 70 C (e.g. transfer the needed vlume int a tube and place it at the apprpriate temperature int a thermmixer) Weigh 200 mg f stl sample (fresh r frzen) int a 2.0 ml Safe-Lck-Tube and add 1.2 ml Lysis Buffer P t each stl sample.vrtex vigrusly fr 1 min. Even if yu use less starting material, perfrm the prtcl like described. Imprtant: If the sample is liquid, pipet 200 µl int the 2.0 ml Safe-Lck-Tube. Cut-ff the end f the pipet tip t make pipetting easier. If the sample is frzen, use a scalpel r spatula t scrape bits f stl int the prvided 2.0 ml Safe-Lck-Tube n ice. Take care, that this samples d nt thaw until Lysis Buffer P is added, therwise the DNA in the sample may degrade. After additin f the buffer, the fllwing steps can be perfrmed at RT r like recmmended. Incubate the sample fr 10 min at RT under cntinuus shaking at 900 rpm. Centrifuge the sample at x g ( rpm) fr 1 min t pellet slid stl particles. Fr an enrichment f bacterial DNA: Incubate the sample fr 10 min at 95 C in a thermmixer under cntinuusly shaking at 900 rpm. Add 5 Zircnia Beads II t the hmgenate and vrtex fr 2 min at RT. Centrifuge the sample at x g ( rpm) fr 1 min t pellet slid stl particles and beads. Imprtant: The incubatin step at 95 C will maximize the yield f bacterial DNA, because f a very efficient disruptin f the cell wall f e.g. gram psitive bacteria. Fr an enrichment f hst DNA, dn t perfrm this high-temperature step 2. Remval f PCR inhibitirs Transfer the supernatant int an InviAdsrb-Tube and vrtex vigrusly fr 15 sec. Incubate the suspensin fr 1 min at rm temperature. Centrifuge the sample at full speed fr 3 min. 17 PSP Spin Stl System 0218

19 PSP Spin Stl DNA Kit 3. Secnd sample cleanup Transfer the supernatant cmpletely int a new 1.5 ml Receiver Tube. Discard the pellet. Centrifuge the sample again at full speed fr 3 min. 4. Prteinase K digestin Transfer 25 µl Prteinase K int a new 2.0 ml Safe-Lck-Tube and pipet 400 µl f the supernatant frm step 3 t the 1.5 ml Receiver Tube cntaining Prteinase K. Mix shrtly by vrtexing and incubate the sample fr 10 min at 70 C in a thermmixer under cntinuus shaking at 900 rpm. 5. Binding f the DNA Add 200 µl Binding Buffer A t the lysate and mix shrtly by vrtexing r by pipetting up and dwn several times. Transfer the mixture cmpletely nt the membrane f the RTA Spin Filter. Incubate fr 1 min at rm temperature and centrifuge at x g ( rpm) fr 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 µl Wash Buffer I t the membrane f the RTA Spin Filter. Clse the lid and centrifuge at x g ( rpm) fr 1 min. Discard the filtrate and the RTA Receiver Tube. Put the RTA Spin Filter in a new RTA Receiver Tube. Add 700 µl Wash Buffer II t the membrane f the RTA Spin Filter. Clse the lid and centrifuge and centrifuge at x g ( rpm) fr 1 min. Discard the filtrate and put the RTA Spin Filter back t the same RTA Receiver Tube. 7. Ethanl remval T eliminate any traces f ethanl, centrifuge again fr 4 min at maximum speed, discard the RTA Receiver Tube 8. DNA Elutin Place the RTA Spin Filter int a new 1.5 ml Receiver Tube and add µl preheated (70 C) Elutin Buffer t the sample. Incubate fr 1 min at RT. Centrifuge at x g ( rpm) fr 1 min. t elute the DNA. Finally discard the RTA Spin Filter. Nte: The DNA can als be eluted with a lwer vlume f Elutin Buffer (depends n the expected yield f genmic DNA). But pay attentin that the minimum vlume fr the elutin is 50 µl and that this vlume can reduce the maximum yield. If a quite large amunt f DNA is expected, the vlume f elutin can be increased. Nte: Fr lng-term strage, we recmmend t keep the eluted DNA at 20 C. 18 PSP Spin Stl System 0218

20 PSP Spin Stl DNA Kit Prtcl 2: Islatin f ttal DNA frm up t 200 mg stl samples frm difficult t lyse bacteria Please read prtcls prir the start f the preparatin and cmplete preparing steps!! Attentin: Please be aware, that yu have t prepare the Binding Buffer A see instructin page: 13 Imprtant Nte: T lyse sme special bacteria cmpletely, (like Mycbacteria paratuberculsis r Chlamydia) a special treatment is necessary. Heat heating blcks (e.g. thermmixer) t 70 C and 95 C Prepare a cntainer with crushed ice 1. Sample hmgenizatin and prelysis Preheat the Elutin Buffer t 70 C (e.g. transfer the needed vlume int a tube and place it at the apprpriate temperature int a thermmixer) Weigh 200 mg f stl sample (fresh r frzen) int a 2.0 ml Safe-Lck-Tube and add 1.2 ml Lysis Buffer P t each stl sample.vrtex vigrusly fr 1 min. Imprtant: If the sample is liquid, pipet 200 µl int the 2.0 ml Safe-Lck-Tube. Cut the end f the pipet tip t make pipetting easier. Incubate the hmgenized sample fr 10 min at 95 C in a thermmixer under cntinuus shaking at 900 rpm. Incubate the sample n ice fr 3 minutes Add 5 Zircnia Beads II t the hmgenate Put the sample back t the 95 C therm blck, Incubate fr further 3 min at 95 C. Vrtex the sample fr 2 min. Centrifuge the sample at x g ( rpm) fr 1 min t pellet slid stl particles. 2. Remval f PCR Inhibitirs Transfer the supernatant int an InviAdsrb-Tube and vrtex vigrusly fr 15 sec. Incubate the suspensin fr 1 min at rm temperature. Centrifuge the sample at full speed fr 3 min. 3. Secnd Sample Cleanup Transfer the supernatant cmpletely int a new 1.5 ml Receiver Tube. Discard the pellet. Centrifuge the sample again at full speed fr 3 min. 4. Prteinase K digestin Transfer 25 µl Prteinase K int a new 2.0 ml Safe-Lck-Tube and pipet 400 µl f the supernatant frm step 3 t the 1.5 ml Receiver Tube cntaining Prteinase K, mix shrtly by vrtexing and incubate the sample fr 10 min at 70 C in a thermmixer under cntinuus shaking at 900 rpm. 5. Binding f the DNA Add 200 µl f Binding Buffer A t the lysate and mix shrtly by vrtexing r by pipetting up and dwn several times. Transfer the mixture cmpletely nt the membrane f the RTA Spin Filter. Incubate fr 1 min at rm temperature and centrifuge at x g ( rpm) fr 2 min. Discard the filtrate and the RTA Receiver Tube. 19 PSP Spin Stl System 0218

21 PSP Spin Stl DNA Kit 6. Washing Steps Put the RTA Spin Filter in a new RTA Receiver Tube. Add 500 µl Wash Buffer I t the membrane f the RTA Spin Filter. Clse the Clse the lid and centrifuge at x g ( rpm) fr 1 min. Discard the filtrate and the RTA Receiver Tube. Put the RTA Spin Filter in a new RTA Receiver Tube. Add 700 µl Wash Buffer II t the membrane f the RTA Spin Filter. Clse the Clse the lid and centrifuge at x g ( rpm) fr 1 min. Discard the filtrate and put the RTA Spin Filter back t the same RTA Receiver Tube. 7. Ethanl Remval T eliminate any traces f ethanl, centrifuge again fr 4 min at maximum speed, discard the RTA Receiver Tube 8. DNA Elutin Place the RTA Spin Filter int a new 1.5 ml Receiver Tube and add µl preheated (70 C) Elutin Buffer. Incubate fr 1 min. Centrifuge at x g ( rpm) fr 1 min. t elute the DNA. Finally discard the RTA Spin Filter. Nte: The DNA can als be eluted with a lwer vlume f Elutin Buffer (depends n the expected yield f genmic DNA). But pay attentin that the minimum vlume fr the elutin is 50 µl and that this vlume can reduce the maximum yield. If a quite large amunt f DNA is expected, the vlume f elutin can be increased. Nte: Fr lng-term strage, we recmmend t keep the eluted DNA at 20 C. 20 PSP Spin Stl System 0218

22 PSP Spin Stl DNA Plus Kit & PSP Spin Stl DNA Basic Kit Scheme f the PSP Spin Stl DNA Plus Kit & PSP Spin Stl DNA Basic Kit (in cmbinatin with the Stl Cllectin Tubes with DNA Stabilizer) Please read prtcls prir the start f the preparatin carefully cllect a spn f the stl sample transfer stl sample int the Stl Cllectin Tube with DNA Stabilizer, clse the tube mix thrughly by shaking r vrtexing t disslve the sample transfer 1.4 ml f the stabilized stl sample (Stl DNA Stabilizer with stl specimen) int a 2.0 ml Safe-Lck Tube fr enrichment f hst DNA: incubate 10 min at RT under shaking fr enrichment f bacterial DNA: incubate 10 min at 95 C n a thermmixer under shaking, add 5 Zircnia Beads II t the hmgenate and vrtex fr 2 min spin dwn at x g ( rpm) fr 1 min transfer the supernatant t the InviAdsrb-Tube mix it by vrtexing fr 15 sec. incubate 1 min at RT spin dwn fr 3 min at full speed. transfer the supernatant in a new 1.5 ml Receiver Tube centrifuge the sample again at full speed fr 3 min add 25 µl Prteinase K in a new 2.0 ml Safe-Lck-Tube transfer 800 µl f the sample supernatant t the same tube mix shrtly by vrtexing incubate fr 10 min at 70 C while cntinuusly shaking n a thermmixer at 900 rpm add 400 µl Binding Buffer A (fllw preparing instructins) t the lysate mix shrtly by vrtexing r pipetting up and dwn transfer the whle mixture in tw steps t the RTA Spin Filter incubate fr 1 min at RT centrifuge at x g ( rpm) fr 2 min discard the filtrate and the RTA Receiver Tube transfer the RTA Spin Filter in a new RTA Receiver Tube pipet 500 µl Wash Buffer I nt the RTA-Spin Filter centrifuge at x g ( rpm) fr 1 min discard the flw-thrugh and the RTA Receiver Tube put the RTA Spin Filter in a new RTA Receiver Tube pipet 700 µl Wash Buffer II nt the RTA Spin Filter centrifuge at x g ( rpm) fr 1 min discard the flw-thrugh and reuse the RTA Receiver Tube t eliminate any traces f ethanl, centrifuge again fr 4 min at maximum speed, discard the RTA Receiver Tube transfer the RTA Spin Filter int a new 1.5 ml Receiver Tube pipet µl f Elutin Buffer (preheated t 70 C) directly nt the center f the membrane f the RTA Spin Filter incubate fr 1 min at RT centrifuge at x g ( rpm) fr 1 min; discard the RTA-Spin Filter; place the eluted ttal DNA immediately in a refrigeratr r stre it at 20 C 21 PSP Spin Stl System 0218

23 PSP Spin Stl DNA Plus Kit & PSP Spin Stl DNA Basic Kit Instructins The fllwing ntes are valid fr the fllwing three prtcls: Nte: The centrifugatin steps were made with the Centrifuge 5415 D frm Eppendrf. The indicated rpm amunts are referring t this centrifuge. Prtcl 1: Cllectin f the stl sample and stabilizatin Please read prtcls prir the start f the preparatin and cmplete preparing steps!! Nte: The Stl Cllectin Tube cntains 8 ml f DNA Stabilizer. That is a new develped buffer frmulatin which enables the prelysis and stabilizatin f the DNA fr at least 3 mnth at ambient temperature. The Stl DNA Stabilizer is very successful even if bacterial pathgens shuld be detected, which are difficult t lyse because f the structure f their cell walls. 1. Open the Stl Cllectin Tube with DNA Stabilizer and cllect a spn f the stl sample. 2. Transfer the spn with the stl sample back int the Stl Cllectin Tube with DNA Stabilizer and clse the tube very tight. 3. Mix thrughly by shaking r vrtexing t disslve the sample. That will lead t hmgenizatin f the stl sample. Imprtant Ntes: The cllected sample can be stred at ambient temperature fr at least 3 mnth. The strage under Stl DNA Stabilizer will lead t a better yield f bacterial pathgens with difficult t lyse cell walls. Strage time has n influence n the quality r the amunt f hst cell DNA. The cllected sample can als be used immediately after cllectin fr the islatin f DNA. The cllected sample can be frzen at 20 C immediately after cllectin r after strage at ambient temperature fr a later use (fr example fr a secnd DNA islatin). Prtcl 2: Islatin f ttal DNA frm 1.4 ml stabilized stl hmgenate with and withut enrichment f bacterial DNA Please read prtcls prir the start f the preparatin and cmplete preparing steps!! Attentin: Please be aware, that yu have t prepare the Binding Buffer A see instructin page: 13 Imprtant Nte: Please nte that the extracted DNA frm stl sample is by the majrity frm bacterial rigin! Heat heating blcks (e.g. thermmixer) t 70 C and 95 C Preheat the Elutin Buffer t 70 C (e.g. transfer the needed vlume int a tube and place it at the apprpriate temperature int a thermmixer) 1. Sample Hmgenizatin and Prelysis Transfer 1.4 ml f the cllected and well hmgenized stl sample (Stl DNA Stabilizer with stl specimen) after strage r directly after cllectin int the 2.0 ml Safe-Lck Tube. Centrifuge the sample at x g ( rpm) fr 1 min t pellet slid stl particles. This will lead t a reduced amunt f extracted ttal DNA, but is nt influencing the amunt f human DNA 22 PSP Spin Stl System 0218