Invisorb Spin Universal Kit User manual
Contents
1. C stratecee molecular User manual Invisorb Spin Universal Kit for simultaneous isolation of genomic and bacterial DNA viral DNA and viral RNA from 200 ul of fresh or frozen plasma serum urine cell free body fluids as well as rinsed liquid from swabs and supernatant from stool suspension or whole blood 100 ul 1050100x00 Mill stRaTEC Molecular GmbH D 13125 Berlin T Instruction for Invisorb Spin Universal Kit The Invisorb Spin Universal Kit is the ideal tool for isolation and purification of higly pure total nucleic acid like human genomic DNA bacterial DNA viral DNA and RNA from 200 ul of fresh or frozen plasma serum urine cell free body fluids as well as rinsed liquid from swabs breast milk pretreated sputum BAL and supernatant from stool suspension or whole blood 100 ul and additionally only for veterinary applications from allantoic fluid or rinse liquid from cloacal or tracheal swabs and from organ abrasions Due to the high purity the isolated DNA RNA is ready to use for a broad panel downstream applications or can be stored at 20 C 80 C for subsequent use The kit is neither validated for the isolation of total RNA from cultured or isolated cells from tissue samples whole blood or urine nor for genomic DNA from tissue blood cards or dried blood stains Compliance with EU Directive 98 79 EC on in vitro medical devices Not for in vitro diagnostic use in countries2. RNA bacterial and human DNA from a diverse range of starting material The procedures are suitable for use with blood plasma or serum either can contain citrate or EDTA no heparin urine swabs and water Samples can be fresh or frozen they should not be frozen and thawed more than once Starting Material Time for preparation 100 ul fresh or frozen blood depending on sample about 60 min per 200 ul fresh or frozen plasma serum urine CSF storage and source extraction supernatant from stool suspensions 200 ul rinsed liquid of swabs pretreated sputum BAL breast milk Additional materials only for veterinarian applications 20 100 ul animal blood 200 ul amniotic fluid 200 ul supernatant from organ abrasions The amount of purified DNA and or RNA in the Invisorb Spin Universal Kit procedures depend on the sample type sample source transport storage age and the virus titer The procedure is designed to avoid sample to sample cross contaminations and allow safe handling of potentially infectious samples The DNA RNA isolation process is based on the interaction of nucleic acids with silica membranes in optimal buffer conditions After a sample specific lysis using Lysis Buffer HLT and Proteinase K optimal binding conditions are adjusted by the addition of Isopropanol The genomic DNA RNA binds to the RTA Spin Filter Subsequent to three washing steps of the membrane bound nucleic acids the nucleic acids are fina
3. 1 minute at 11 100 x g 11 000 rpm Discard the RTA Receiver Tube with filtrate and place the RTA Spin Filter into a new RTA Receiver Tube 3 First washing of the RTA Spin Filter Add 600 ul Wash Buffer HLT to the RTA Spin Filter and centrifuge at 11 100 x g 11 000 rpm for 1 min Discard the RTA Receiver Tube with filtrate and place the RTA Spin Filter into a new RTA Receiver Tube 4 Second washing of the RTA Spin Filter Add 700 ul Wash Buffer to the RTA Spin Filter and centrifuge at 11 100 x g 11 000 rpm for 1 min Discard the filtrate and put the RTA Spin Filter back into the used RTA Receiver Tube Repeat this washing step once 5 Ethanol removal Remove the residual ethanol by final centrifugation for 5 min at 11 100 x g 11 000 rpm Discard the RTA Receiver Tube with filtrate 6 Elution of the DNA RNA Place the RTA Spin Filter into a 1 5 ml Elution Tube and add 100 200 ul of the Elution Buffer M prewarmed to 65 C directly onto the RTA Spin Filter surface Incubate for 1 min at RT and centrifuge at 11 100 x g 11 000 rpm for 1 minute Discard the RTA Spin Filter Close the 1 5 ml Receiver Tube and store the sample at 20 C to 80 C 20 Invisorb Spin Universal Kit Version 0515 Additional protocol Simultaneous isolation of nucleic acids from tissue biopsies Please read the protocols carefully prior to the start of the preparation procedure Important Note Prewarm the needed amount of Elution Buffer
4. 2 10 Extractions from supernatant from organ abrasions Transfer 100 200 mg tissue material into a 2 0 ml Safe Lock Tube not provided Add 800 ul physiological saline solutions to the material and disrupt the material by using a mill or shredder e g Fastprep from MP Biomedical After the homogenization process wait 10 min for sedimentation of the bigger particles Transfer 200 ul of the cleared supernatant into a 2 0 ml Safe Lock Tube and follow step 1 for NA extraction from bacteria or follow step 2 for NA extractions from virus in protocol 2 13 Invisorb Spin Universal Kit Version 0515 Lysis procedures For easier handling we recommend to prepare a Master Mix only for the needed amount of samples consisting of Lysis Buffer HLT Proteinase K and if required Carrier RNA When preparing the Master Mix it is recommended to use a volume of 5 greater than that required The Master Mix is stable for at least 2h at RT Preparation of a Master Mix Number of Amount of Amount of Amount of samples Lysis Buffer HLT Carrier RNA Proteinase K 200 ul sample 20 ul sample 20 ul sample 6 1 3 ml 130 ul 130 ul 8 1 7 ml 170 ul 170 ul 10 2 1 ml 210 ul 210 ul 12 2 6 ml 260 ul 260 ul 16 3 4 ml 340 ul 340 ul 20 4 2 ml 420 ul 420 ul 24 5 0 ml 500 ul 500 ul 32 6 7 ml 670 ul 670 ul 40 8 4 ml 840 ul 840 ul 48 10 0 ml 1000 ul 1000 ul Extraction control Extraction control DNA or RNA must be
5. M to 65 C for the final elution step Switch on heating blocks e g thermomixer to 65 C and 95 C 1 Sample Lysis Transfer 1 10 mg from the tissue biopsy sample into a 2 0 ml Safe Lock Tube and add 200 ul distilled water or PBS 200 ul Lysis Buffer HLT 20 ul Carrier RNA and 20 ul Proteinase K to each sample Note If you handle more than 5 preps at the same time we suggest preparing a Master Mix as described at point Lysis Procedure page 14 Add 240 ul Master Mix to each sample instead of Carrier RNA Lysis Buffer HLT and Proteinase K The addition of Carrier RNA to the sample is here optional Place the Tube into a thermomixer and incubate under continuous shaking for 10 minutes at 65 C and then for 10 minutes at 95 C Lysis times may be increased if the lysis is not completely For Bacterial DNA Before you add Lysozym to the mixture take care that the sample is cooled down to lt 40 C Add 20 ul Lysozyme stock 10 mg ml not provided to the lysed sample and incubate for 10 min under shaking at RT Important A longer lysis time could be reduce the final yield and quality of some viral RNA species After lysis centrifuge the sample at max speed for 1 minute to spin down unlysed material Transfer the cleared supernatant completely into a 1 5 ml reaction tube not provided 2 Binding of the DNA and RNA Add 260 ul Binding Solution to the 1 5 ml reaction tube and mix the sample completely by pipetting up and d
6. NaOH 1 mM EDTA followed by RNase free water You can also take chloroform resistant plastic ware rinsed with chloroform to inactivate RNases All buffers must be prepared from DEPC treated RNase free ddH20 Change gloves frequently and keep tubes closed Reduce the preparation time as much as possible Use only sterile disposable polypropylene tubes throughout the procedure These tubes are generally RNase free o Keep isolated RNA on ice OO Oo This kit should only be used by personnel trained in laboratory practice Storage of RNA Purified RNA can be stored 80 C and is stable for months and years e g precipitated and stored in 70 ethanol Quantification Quantification of DNA and RNA from this assay must be done by means of qPCR or Reverse Transcriptase qPCR All other methods will be disturbed by the included Carrier Nucleic Acids as well as DNA or RNA which is co purified 25 Invisorb Spin Universal Kit Version 0515 Ordering information Product Package Size Catalogue No Invisorb Spin Universal Kit Kit 50 preparations 1050100200 Invisorb Spin Universal Kit Kit 250 preparations 1050100300 Related Products Package Size Catalogue No InviMag Universal Kit KF96 1 x 96 preparations 7450300100 InviMag Universal Kit KF96 5 x 96 preparations 7450300200 InviMag Universal Kit STARIet 4 x 96 preparations 7450330300 InviMag Universal Kit STARIet 24 x 96 preparations 7450330400 Invisorb
7. RNA is eluted in Elution Buffer M O O OO Lysis Samples are lysed at elevated temperatures in the presence of Lysis Buffer HLT and Proteinase K For bacteria we recommend a pretreatment with Lysozyme at 37 C before lysis Binding nucleic acids After adding Isopropanol to adjust optimal binding conditions the lysate will be applied onto the RTA Spin Filter and the nucleic acids are bound to the surface of the Filter membrane as the lysate is drawn through by centrifugation Removing residual contaminants Contaminants are efficiently washed away using Wash Buffer HLT and Wash Buffer while the nucleic acids remain bound to the membrane of the RTA Spin Filter Elution High quality viral DNA RNA and genomic DNA is eluted from the membrane using Elution Buffer M Eluting twice each time with 100 ul leads to a little increase of DNA RNA yield Usage of small elution volumes may raise the DNA RNA concentration Elution volumes should be at least 40 ul of Elution Buffer M The volume of eluate recovered may be up to 5 ul less than the volume of elution buffer applied to the RTA Spin Filter The volume of eluate recovered depends on the nature of the sample and the amount of Elution Buffer M used The eluted DNA RNA are ready to use in different subsequent applications 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
8. Universal HTS 96 Kit STARIet 4 x 96 preparations 7150330300 Invisorb Universal HTS 96 Kit STARIet 24 x 96 preparations 7150330400 InviMag Universal Kit IG 8 x 12 preps 2450120100 InviMag Pathogen Kit KF96 1 x 96 preparations 7450300100 InviMag Pathogen Kit KF96 5 x 96 preparations 7450300200 InviMag Pathogen Kit KEmL 15 preparations 2450110200 InviMag Pathogen Kit KEmL 75 preparations 2450110300 RTP Pathogen Kit 50 preparations 1040500200 RTP Pathogen Kit 250 preparations 1040500300 RTP DNA RNA Virus Mini Kit 50 preparations 1040100200 RTP DNA RNA Virus Mini Kit 250 preparations 1040100300 RTP Bacteria DNA Mini Kit 50 preparations 1033200200 RTP Bacteria DNA Mini Kit 250 preparations 1033200300 Invisorb Virus RNA HTS 96 Kit X 4 x 96 preparations 7143310300 Invisorb Virus RNA HTS 96 Kit X 24 x 96 preparations 7143310400 Invisorb Virus DNA HTS 96 Kit X 4 x 96 preparations 7142310300 Invisorb Virus DNA HTS 96 Kit X 4 x 96 preparations 7142310400 InviMag Virus RNA Mini Kit KF 96 1 x 96 preparations 7443300100 InviMag Virus RNA Mini Kit KF 96 5 x 96 preparations 7443300200 InviMag Virus DNA Mini Kit KF 96 1 x 96 preparations 7442300100 InviMag Virus DNA Mini Kit KF 96 5 x 96 preparations 7442300200 InviMag Virus DNA RNA Mini Kit KFmL 15 preparations 2441150100 InviMag Virus DNA RNA Mini Kit KFmL 75 preparations 2441150200 InviMag Virus DNA RNA Mini Kit 50 preparations 14401002
9. a thermomixer and incubate under continuous shaking for 10 minutes at 65 C and then for 10 minutes at 95 C Before you add Lysozym to the mixture take care that the sample is cooled down to lt 40 C Add 20 ul Lysozyme stock 10 mg ml not provided to the lysed sample and incubate for 10 min under shaking at RT 1c Sample Lysis for virus NA In a 2 ml Safe Lock Tube mix 200 ul of the sample with 200 ul Lysis Buffer HLT 20 ul Carrier RNA and 20 ul Proteinase K Note If you handle more than 5 preps at the same time we suggest preparing a Master Mix as described at point Lysis Procedure page 14 Add 240 ul Master Mix to each sample instead of Carrier RNA Lysis Buffer HLT and Proteinase K Vortex the sample vigorously for 10 seconds Place the Tube into a thermomixer and incubate under continuous shaking for 10 minutes at 65 C and then for 10 minutes at 95 C Note stool samples These samples have to be diluted 1 10 with RNAse free water Vortex the sample for 30 sec Centrifuge the sample for 1 min at 12 000 rpm and transfer the supernatant in a new tube not provided 19 Invisorb Spin Universal Kit Version 0515 2 Binding of the DNA and RNA Add 260 ul Binding Solution to the lysed sample and mix the sample completely by pipetting up and down or by vortexing Incubate the sample at room temperature for 5 minutes Transfer the sample into the RTA Spin Filter put into a RTA Receiver Tube Close the cap and centrifuge for
10. min at 11 100 x g 11 000 rpm Discard the filtrate and the RTA Receiver Tube Transfer the RTA Spin Filter in a new RTA Receiver Tube Add 600 ul Wash Buffer HLT Centrifuge for 1 min at 11 100 x g 11 000 rpm Discard the filtrate and the Receiver Tube Place the RTA Spin Filter into a new RTA Receiver Tube Add 700 ul Wash Buffer Centrifuge for 1 min at 11 100 x g 11 000 rpm Discard the filtrate and put the RTA Spin Filter back into the used RTA Receiver Tube repeat this washing step once centrifuge for 5 min at 11 100 x g 11 000 rpm Discard the RTA Receiver Tube with filtrate Place the RTA Spin Filter into a 1 5 ml Receiver Tube Add 100 200 ul of Elution Buffer M preheated to 65 C Incubate for 1 min at room temperature Centrifuge for 1 min at 11 100 x g 11 000 rpm Discard the RTA Spin Filter Close the 1 5 ml Receiver Tube and store the DNA sample at 20 C to 80 C C 18 Invisorb Spin Universal Kit Version 0515 Protocol 2 Simultaneous isolation of pathogen DNA and RNA from all liquid samples Please read the protocols carefully prior to the start of the preparation procedure Important Note Prewarm the needed amount of Elution Buffer M to 65 C for the final elution step The protocol has been optimized for the isolation of total nucleic acids from body fluids of 200 ul blood 100 ul For samples which have a smaller volume than 200 ul please fill up to a total volume of 200 ul with Elution buffer M 1a
11. samples is stable for months Viral RNA purification should be processed as soon as possible Samples can also be stored in the dissolved Lysis Buffer in the Extraction Tube L for 1 h at room temperature overnight at 4 C and for long term storage at 80 C Storage under deep frozen conditions is recommended Biopsy material tissue Best results are obtained with fresh material or material that has been immediately frozen and stored at 20 C or 80 C Repeated freezing and thawing of stored samples should be avoided since this leads to reduced DNA size Use of poor quality starting material also leads to reduced length and influences yield of purified DNA The amount of purified DNA from max 10 mg tissue sample depends on the nature of starting material The thawing process could proceed Cultivated bacterial Bacteria have to be pelleted after cultivation Best results are obtained with fresh material or material that has been immediately frozen and stored at 20 C or 80 C Repeated freezing and thawing of stored samples should be avoided since this leads to reduced DNA size Processing of bacterial samples The kit was validated with Bacillus subtilis spiked cell free medium To perform a quantitative extraction of bacterial DNA from gram positive Bacteria addition of Lysozyme is needed Please add 5 ul of a 10 mg ml Lysozyme solution per 200 ul sample volume to the primary tube before starting the assay Urine The bacteria mu
12. step at 12 000 x g 13 000 rpm Transfer 200 ul virus containing supernatant into a 2 0 ml Safe Lock Tube prevent the aspiration of swimming particles and follow step 2 in protocol 2 6 Extraction of bacterial NA from supernatant of stool suspension Transfer 100 ul 100 mg stool sample into a 2 ml tube and add 300 ul RNase free Water Vortex the sample for 30 s followed by a 30 s centrifugation step at 3 000 rpm 1 000 x g Transfer 200 ul of the bacteria containing supernatant into a 2 0 ml Safe Lock Tube prevent the aspiration of swimming particles and follow step 1 in protocol 2 7 Extraction of NA from Biopsy material tissue Transfer 1 10 mg from the tissue biopsy sample into tube and add 200 ul distilled water 200 ul Lysis Buffer HLT 20 ul Carrier RNA and 20 ul Proteinase K to the sample If genomic DNA shall be prepared the addition of Carrier RNA to the Master Mix or to the sample is optional 8 Extraction from bacterial culture Transfer 1ml of the bacterial culture into a 2 0 ml Safe Lock Tube not provided Centrifuge the overnight culture for 2 min at 8000 rpm and remove completely the supernatant Resuspend the bacteria pellet in 200 ul PBS Buffer not provided and follow step 1 in protocol 2 9 Extraction from amniotic fluid Open the infected egg after cultivation for 5 7 days at 37 C depending on the virus Transfer 200 ul of the allantois liquid into a 2 0 ml Safe Lock Tube and follow step 2 in protocol
13. 00 2 0 ml reaction tubes optional Measuring cylinder 250 ml Disposable gloves Pipet with tips 1 5 ml reaction tubes 10 optional centrifuge for 15 or 50 ml 11 optional Lysozyme 10 mg ml 12 optional PBS O00 NI 0 O SoS The Invisorb Spin Universal Kit is validated with 2 Propanol Rotipuran gt 99 7 p a ACS ISO Order no 6752 from Carl Roth Possible suppliers for lsopropanol Carl Roth Applichem Sigma 2 Propanol 2 Propanol f r die Molekularbiologie 2 Propanol Rotipuran gt 99 7 p a ACS ISO Order no A3928 Order no 59304 1L F Order no 6752 Possible suppliers for centrifuges Eppendorf AG SIGMA Laborzentrifugen GmbH 22331 Hamburg Germany 37507 Osterode am Harz Germany Tel 49 0 40 53801 0 Tel 49 5522 5007 0 Fax 49 0 40 53801 556 Fax 49 5522 5007 12 E Mail eppendorf eppendorf com E Mail info sigma zentrifugen de Internet www eppendorf com Internet www sigma zentrifugen de Important indications Carrier RNA Carrier RNA serves the following purposes It enhances the reversible binding of viral acids to the RTA Spin Filter membrane The addition of Carrier RNA reduces the chance of viral nucleic acid degradation It minimizes the binding of viral acid to the reaction tubes Handling of RTA Spin Filter Do to the sensitivity of viral DNA RNA amplification technologies the following precautions are necessary when handling the RTA Spin Filter to avoid cross contamination between
14. 00 InviMag Virus DNA RNA Mini Kit 250 preparations 1440100300 InviMag Bacteria DNA Mini Kit KFmL 15 preparations 2433150100 InviMag Bacteria DNA Mini Kit KFmL 75 preparations 2433150200 Possible suppliers for lsopropanol Carl Roth Applichem Sigma 2 Propanol 2 Propanol 2 Propanol Rotipuran gt 99 7 p a ACS ISO Order Nr A3928 Order Nr 59304 1L F Order Nr 6752 Possible suppliers for centrifuges Eppendorf AG 22331 Hamburg Germany Tel 49 0 40 53801 0 Fax 49 0 40 53801 556 E Mail eppendorf eppendorf com Internet www eppendorf com SIGMA Laborzentrifugen GmbH 37507 Osterode am Harz Germany Tel 49 5522 5007 0 Fax 49 5522 5007 12 E Mail info sigma zentrifugen de Internet www sigma zentrifugen de 26 Invisorb Spin Universal Kit Version 0515 stratecee molecular STRATEC Molecular GMbH Robert R ssle 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 1A8k 05 2015
15. IANS 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 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 isolation of eukaryotic or total RNA from any kind of sample was not evaluated The isolation of DNA RNA from sample sources like fungi was neither tested nor validated Differing the starting material or flow trace may lead to inoperability Therefore neither a warranty nor a guarantee in this case will be given implied or expressed The user is responsible to validate the performance of the STRATEC Molecular product for any particular use STRATEC Molecular does not provide validations of performance characteristics of the product with respect to specific applications STRATEC Molecular products may be used in clinical diagnostic laboratory systems c
16. Sample Lysis for bacterial NA Note For parallel isolation of bacterial DNA and viral RNA please use Lysis Protoocl 1b Note Bacterial culture centrifuge max 0 5 ml of an overnight culture for 2 min at 8000 rpm and remove completely the supernatant Resuspend the bacteria pellet in 200 ul PBS Buffer not provided In a 2 ml Safe Lock Tube mix 200 ul of the sample with 20 ul Lysozym mixture 10 mg ml and 20 ul Carrier RNA Mix vigorously by vortexing Incubate for 10 min at 37 C Add 200 ul Lysis Buffer HLT and 20 ul Proteinase K Note If you handle more than 5 preps at the same time we suggest preparing a Master Mix as described at point Lysis Procedure page 14 Add 240 ul Master Mix to each sample instead of Carrier RNA Lysis Buffer HLT and Proteinase K Vortex the sample vigorously for 10 seconds Place the Tubes into a thermomixer and incubate under continuously shaking for 10 to 15 minutes at 65 C and then optional for 10 min at 95 C 1b Sample Lysis for simultaneous isolation of bacterial NA and viral NA In a 2 ml Safe Lock Tube mix 200 ul of the sample with 200 ul Lysis Buffer HLT 20 ul Carrier RNA and 20 ul Proteinase K Note If you handle more than 5 preps at the same time we suggest preparing a Master Mix as described at point Lysis Procedure page 14 Add 240 ul Master Mix to each sample instead of Carrier RNA Lysis Buffer HLT and Proteinase K Vortex the sample vigorously for 10 seconds Place the Tube into
17. able cause Proteinase K volume concentration too low reagent buffer volume too low no too much isopropanol added to Wash Buffer HLT no too much ethanol added to Wash Buffers incorrect storage of starting material old material combination of reagents from different kits 23 Comments and suggestions make sure that you have resuspended the lyophilized Proteinase K with the appropriate volume of water before use ensure that the Wash Buffer HLT has been filled up with isopropanol properly as indicated in Tab 1 ensure that the Wash Buffer has been filled up with ethanol properly as indicated in Tab 1 ensure that the storage of starting material was correct avoid multiple freezing and thawing cycles of the material ensure that the starting material is fresh or stored under appropriate condition for long time storage at 20 C avoid multiple thawing and freezing cycles of the material old material often contains degraded DNA RNA please make sure that only reagents belonging to one kit type are used a combination of reagents belonging to different kit types will not work Invisorb Spin Universal Kit Version 0515 Appendix General notes on handling DNA Starting material This kit is designed for extraction of DNA from saliva and swabs These materials show big variation in DNA contents The purification of some apoptotic DNA is normal Nature of DNA The length and delicate physica
18. and RNA from all liquid samples and from tissue biopsies 18 Protocol 2 Simultaneous isolation of pathogen DNA and RNA from all liquid samples 19 Additional protocol Simultaneous isolation of nucleic acids from tissue biopsies 21 Troubleshooting 23 Appendix 24 Ordering information 26 2 Invisorb Spin Universal Kit Version 0515 Kit contents of the Invisorb Spin Universal Kit 10 Preparations 50 Preparations 250 Preparations Catalogue No 1050100900 1050100200 1050100300 Lysis Buffer HLT 2x2 ml 15 ml 60 ml Proteinase K for 250 Hl for 1 1 ml for 3x2 ml working solution working solution working solution Carrier RNA for 240 Hl for 1 2 ml for 3 x 2 ml working solution working solution working solution RNase Free Water 2 ml 2x2ml 15 ml Binding Solution 3x1 ml empty bottle empty bottle fill with 99 7 Isopropanol ready to use final volume 15 ml final volume 80 ml Wash Buffer HLT 15 ml 30 ml 105 ml ready to use final Volume 50 ml final Volume 175 ml Wash Buffer 15 ml 2x18 ml 2 x 60 ml ready to use final Volume 2 x 60 ml final Volume 2 x 200 ml Elution Buffer M 2x2 ml 30 ml 120 ml RTA Spin Filter Set 10 50 5 x 50 RTA Receiver Tubes 20 2x 50 10 x 50 1 5 ml Receiver Tubes 10 50 5 x 50 2 0 ml Safe Lock Tubes 10 50 5 x 50 Manual 1 1 1 Initial steps Resuspend Resuspend lyophillized Resuspend lyophillized lyophillized Proteinase K by addition of Proteinase K by addition of Pr
19. combined with the provided Carrier RNA in one mixture The vials with Carrier RNA contain 240 ul 1 2 ml or 2 0 ml stock solutions depending on the package size Add the respective amount of Extraction Control Nucleic Acid to the Carrier RNA if it is in a bigger volume gt 25 you may replace the according amount of RNAse free water Notes If you only have indication of amount per reaction please calculate by using eluate and template volume If the extraction control is stable in plasma serum CSF urine respiratory samples whole blood stool transport media or on dried swabs e g armored RNA it can alternatively be added to the sample shortly before beginning sample preparation If the extraction control is naked DNA or RNA it is unstable in plasma serum CSF urine respiratory samples whole blood stool transport media or on dried swabs and must not be added directly to the samples In carrier RNA an extraction control stabilized Refer to the manufacturer s instructions to determine the optimal amount of extraction control for specific downstream applications Using an amount other than that recommended may lead to wrong quantification results 14 Invisorb Spin Universal Kit Version 0515 Instructions The following notes are valid for all protocols Note The DNA RNA can also be eluted with a lower but not lower than 40 ul or a higher volume of Elution Buffer M depends on the expected yield or needed co
20. 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 liquid spillage refer to Safety Information see page 6 Do not use damaged kit components since their use may lead to poor kit performance o Always change pipette tips between liquid transfers To avoid cross contamination we recommend the use of aerosol barrier pipette tips o When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles Discard gloves if they become contaminated 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 o This kit should only be used by trained personnel O Or OO 8 Invisorb Spin Universal Kit Version 0515 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 gt 11 000 x g 11 000 rpm Thermomixer 37 C 95 C Isopropanol 99 7 Ethanol 96 1
21. de any warranty for efficiency of the used cultivation method b the sample will not be used for cultivation Cut off the relevant part of the swab and transfer this part into an RNase and DNAse free 2 ml tube Add 400 ul RNase free water to the swab and vortex intensely for 3 min Optional incubate for 3 min at 95 C Transfer 200 ul of the rinsed liquid into a 2 0 ml Safe Lock Tube and follow step 1 for NA extraction from bacteria or follow step 2 for NA extractions from virus in protocol 2 optional If bacterial DNA is processed 20 ul Lysozyme can be added to 200 ul sample follow the instructions of protocol 2 3 Extraction of NA from sputum Transfer 200 ul from the sputum sample into an RNase DNAse free tube and add 200 ul NAC Buffer order number 1033221100 ratio sample to buffer must be 1 1 or add 400 ul saturated Acetylcysteine ACC solution to the sample ratio sample to buffer must be 1 2 Incubate the mixture for 10 min at 95 C under shaking on the thermomixer to reduce the viscosity and transfer 200 ul from the mixture into a 2 0 ml Safe Lock Tube and follow step 1 for NA extraction from bacteria or follow step 2 for NA extractions from virus in protocol 2 4 Extraction of NA from slimy tracheal secretes or BAL Non viscous samples For isolation of bacterial DNA transfer 1 ml of tracheal secret or BAL into a RNase DNAse free tube and centrifuge at 11 100 x g 11 000 rpm for 3 min Discard decant the supernatant withou
22. eiver Tube repeat this washing step once centrifuge for 5 min at 11 100 x g 11 000 rpm Discard the RTA Receiver Tube with filtrate Place the RTA Spin Filter into a 1 5 ml Receiver Tube Add 100 200 ul of Elution Buffer M preheated to 56 C Incubate for 1 min at room temperature Centrifuge for 1 min at 11 100 x g 11 000 rpm genomic DNA Discard the RTA Spin Filter Close the 1 5 ml Receiver Tube and store the DNA sample at 4 C for long term storage at 20 C to 80 C 16 Invisorb Spin Universal Kit Version 0515 Protocol 1 Isolation of genomic DNA from blood Please read the protocols carefully prior to the start of the preparation procedure Important Note Prewarm the needed amount of Elution Buffer M to 56 C for the final elution step The protocol has been optimized for the isolation of total nucleic acids from 100 ul blood or other body fluids of 200 ul For samples which have a smaller volume than 200 ul please fill up to a total volume of 200 ul with Elution Buffer M 1 Sample Lysis In a 2 ml Safe Lock Tube mix 100 ul of the blood sample with 100 ul Elution Buffer M add 200 ul Lysis Buffer HLT and 20 ul Proteinase K Vortex the sample vigorously for 10 seconds Place the Tubes into a thermomixer and incubate under continuously shaking for 15 minutes at 56 C Attention by using animal blood the amount of starting material can vary between 20 and 100 ul Before starting using the kit make please a d
23. he RTA Spin Filter Close the 1 5 ml Receiver Tube and store the DNA sample at 4 C for long term storage at 20 C to 80 C 17 Invisorb Spin Universal Kit Version 0515 Scheme for the simultaneous isolation of pathogen DNA and RNA from all liquid samples Pathogen NA Please read protocols prior the start of the preparation carefully Transfer 200 ul of origin or pretreated sample into a 2 0 ml Safe Lock Tube For samples which have a smaller volume than 200 ul please adjust to a total volume of 200 ul with Elution Buffer M Only for isolation of bacterial DNA Add 20 ul Lysozyme and 20 ul Carrier RNA and mix vigorously by vortexing Incubate for 10 min at 37 C Add 200 ul Lysis Buffer HLT and 20 ul Proteinase K vortex vigorously Only for isolation of viral DNA and RNA Add 200 ul Lysis Buffer HLT 20 pl Carrier RNA and 20 ul Proteinase K vortex vigorously Note If you handle more than 5 preps at the same time we suggest preparing a Master Mix as described at point Lysis Procedure page 14 Add 240 ul Master Mix to each sample instead of Carrier RNA Lysis Buffer HLT and Proteinase K For all Incubate for 10 min at 65 C and then for 10 min at 95 C while continuously shaking Add 260 ul Binding Solution and mix by pipetting up and down four times or vortexing Incubate the sample at room temperature for 5 minutes Take a RTA Spin Filter System Transfer lysate onto the RTA Spin Filter Centrifuge for 1
24. ilution series and detect the optimal amount 2 Binding of the DNA and RNA Add 260 ul Binding Solution to the lysed sample and mix the sample completely by pipetting up and down or by vortexing Incubate the sample at room temperature for 5 minutes Transfer the sample into the RTA Spin Filter put in a RTA Receiver Tube Close the cap and centrifuge for 1 minute at 11 100 x g 11 000 rpm Discard the RTA Receiver Tube with filtrate and place the RTA Spin Filter into a new RTA Receiver Tube 3 First Washing of the RTA Spin Filter Add 600 ul Wash Buffer HLT to the RTA Spin Filter and centrifuge at 11 100 x g 11 000 rpm for 1 min Discard the RTA Receiver Tube with filtrate and place the RTA Spin Filter into a new RTA Receiver Tube 4 Second Washing of the RTA Spin Filter Add 700 ul Wash Buffer to the RTA Spin Filter and centrifuge at 11 100 x g 11 000 rpm for 1 min Discard the filtrate and put the RTA Spin Filter back into the used RTA Receiver Tube Repeat this washing step once Ethanol removal Remove the residual ethanol by final centrifugation for 5 min at 11 100 x g 11 000 rpm Discard the RTA Receiver Tube with filtrate 7 Elution of the DNA RNA Place the RTA Spin Filter into a 1 5 ml Elution Tube and add 100 200 ul of the Elution Buffer M prewarmed to 56 C directly onto the RTA Spin Filter surface 8 Incubate for 1 min at room temperature and centrifuge at 11 100 x g 11 000 rpm for 1 minute Discard t
25. l infectious materials Contamination of the waste with residual infectious materials is unlikely but cannot be excluded completely Therefore the waste has to be considered infectious and should be handled and discarded according to local safety regulations European Community risk and safety phrases for the components of the Invisorb Spin Universal Kit to which they apply are listed below as follows Lysis Buffer HLT Wash Buffer HLT Proteinase K waming danger contains guanidine hydrochloride H302 315 319 P280 305 351 338 H315 319 334 335 P280 305 35 1 338 310 405 H315 Causes skin irritation H319 Causes serious eye irritation H334 May cause allergy or asthma symptoms or breathing difficulties if inhaled H335 May cause respiratory irritation P280 Wear protective gloves protective clothing eye protection face protection P305 P351 P338 If in eyes Rinse cautiously with water for several minutes Remove contact lenses and continue rinsing P310 Immediately call a POISON CENTER or doctor physician P405 Store locked up Emergency medical information can be obtained 24 hours a day from infotrac outside of USA 1 352 323 3500 inside of USA 1 800 535 5053 6 Invisorb Spin Universal Kit Version 0515 Product characteristics of the Invisorb Spin Universal Kit The Invisorb Spin Universal Kit provides a fast and efficient way for reliable simultaneous isolation of high quality viral DNA
26. l nature of DNA requires 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 compatibility with various downstream applications Damaged DNA could perform poorly in applications such as genomic Southern blotting and long template PCR Storage of DNA A working stock of DNA can be stored at 2 4 C for several weeks For long term storage DNA should be stored at 20 C but storing at 20 C can cause shearing particularly if the DNA is exposed to repeated freeze thaw cycles Note that the solution in which the nucleic acid is eluted in will affect it s stability during storage Pure water lacks buffering capacity and an acidic pH may lead to acid hydrolysis Tris or Tris EDTA buffer contains sufficient buffering capacity to prevent acid hydrolysis Drying dissolving and pipetting DNA Avoid over drying 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 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 24 Invisorb Spin Unive
27. lly eluted in Elution Buffer M Yield and quality of the isolated nucleic acids are suitable for a wide range of molecular diagnostic detection system The diagnostic tests should be performed according to manufacturer specifications Due to the high purity the isolated DNA RNA is ready to use for a broad panel of downstream applications or can be stored at 80 C for subsequent use RT PCR real time PCR quantitative RT PCR like TaqMan und LightCycler technology cDNA synthesis microarray application OoO00 0 Note Systems isolating simultaneously DNA and RNA using buffers adapted for the binding of DNA and RNA but the optimal binding conditions of RNA and DNA are different so that such solutions can show a little reduction in sensitivity in comparison to kits optimized to one kind of nucleic acid isolation For further information please contact 49 0 30 9489 2901 2910 in Germany and from foreign countries 49 0 30 9489 2907 2903 or your local distributor see page 28 7 Invisorb Spin Universal Kit Version 0515 Principle and procedure The Invisorb Spin Universal Kit procedure comprises following steps Lysis and protein digestion Binding of the DNA RNA to the filter membrane Washing the filter bound DNA RNA and elimination of alcohol Elution of DNA RNA After lysis the DNA RNA binds to the filter contaminations and enzyme inhibitors are efficiently removed during the following wash steps and purified DNA
28. ls Lot number Catalogue number Expiry date Consult operating instructions Temperature limitation Do not reuse E roa Manufacturer Storage All buffers and kit contents of the Invisorb Spin Universal Kit should be stored at room temperature and are stable for at least 12 months Room temperature RT is defined as range from 15 30 C Proteinase K Dissolved Proteinase K must be stored at 20 C Dividing the Proteinase K into aliquots and storage at 20 C is recommended Carrier RNA Lyophilized Carrier RNA can be stored at 2 8 C or at RT but the recommendation for long time storage is 20 C Dissolved Carrier RNA must be stored at 80 C but repeated freezing and thawing will degrade the Carrier RNA and reduce the functionality of the kit Therefore dividing Carrier RNA into aliquots and storage at 80 C is recommended Wash Buffers Wash Buffer charged with isopropanol or ethanol should be stored at room temperature and should be appropriately sealed If any precipitates are visible within the provided solutions solve these precipitates by careful warming up to 30 C Quality control and product warranty STRATEC Molecular warrants the correct function of the Invisorb Spin Universal Kit for applications as described in this manual Purchaser must determine the suitability of the Product for its particular use Should any Product fail to perform the applications as described in the manual STRATEC Molec
29. n be stored on ice for 1 2 hours for short time up to 24 h samples may be stored at 20 C For long term storage we recommend freezing samples in aliquots at 80 C Frozen plasma or serum samples must not be thawed more than once Multiple thawing and freezing before isolating the viral DNA RNA should be avoided It leads to denaturation and precipitation of proteins resulting in reduced viral titers and therefore reduced yields of viral nucleic acids In addition cryoprecipitate formed during freeze thawing could make problems If cryoprecipitate is visible they should be pelleted by centrifugation at app 6 800 x g for 3 minutes The cleared supernatant should be aspirated without disturbing the pellet and processed immediately This step will not reduce viral titers Swabs saliva The protocol works with fresh saliva prepared swabs as well as with dried swabs The protocol has not been validated for isolation of DNA from swabs which are stored under special storage buffers of other provider 10 Invisorb Spin Universal Kit Version 0515 Best results are obtained using freshly extracted samples As long as the samples are not shock frosted with liquid nitrogen or are incubated with RNase inhibitors or denaturing reagents the viral RNA is not secured Therefore it is essential that samples are immediately flash frozen subsequent to the harvesting by using liquid nitrogen and are stored at 80 C Viral RNA contained in such deep frozen
30. ncentration of the DNA RNA The eluate can contain viral DNA viral RNA bacterial or genomic DNA Important After extraction place the Elution Tube on ice For long time storage place the nucleic acids at 20 C or 80 C Note The centrifugation steps were made with the Centrifuge 5415 D from Eppendorf The indicated rpm amounts are referring to this centrifuge 15 Invisorb Spin Universal Kit Version 0515 Scheme for the Isolation of genomic DNA from blood Please read protocols prior the start of the preparation carefully Transfer 100 ul of blood sample into a 2 0 ml Safe Lock Tube and add 100 ul Elution Buffer M Add 200 ul Lysis Buffer HLT and 20 ul Proteinase K vortex vigorously Incubate for 15 min at 56 C while continuously shaking Add 260 ul Binding Solution and mix by pipetting up and down four times or vortexing Incubate the sample at room temperature for 5 minutes Take a RTA Spin Filter Set Transfer lysate onto the RTA Spin Filter Centrifuge for 1 min at 11 100 x g 11 000 rpm Discard the filtrate and the RTA Receiver Tube Transfer the RTA Spin Filter in a new RTA Receiver Tube Add 600 ul Wash Buffer HLT Centrifuge for 1 min at 11 100 x g 11 000 rpm Discard the filtrate and the RTA Receiver Tube Place the RTA Spin Filter into a new RTA Receiver Tube Add 700 ul Wash Buffer Centrifuge for 1 min at 11 100 x g 11 000 rpm Discard the filtrate and put the RTA Spin Filter back into the used RTA Rec
31. onditioned upon the complete diagnostic system of the laboratory The laboratory must be validated pursuant to CLIA 88 regulations in the U S or equivalents in other countries 5 Invisorb Spin Universal Kit Version 0515 All products sold by STRATEC Molecular are subject 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 other purposes than intended The included chemicals are only useable once and are not suitable for consumption Safety information When and while working with chemicals always wear a suitable lab coat disposable gloves and protective goggles Avoid skin contact Adhere to 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 Product and its components If 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 waste generated by the Invisorb Spin Universal Kit procedures for residua
32. oteinase K by addition of 250 ul RNase free Water to the vial and mix thoroughly until completely dissolving and store at 20 C Resuspend lyophillized Carrier RNA by addition of 240 ul RNase free Water to the vial and mix thoroughly until completely dissolving 1 1 ml RNase free Water to the vial mix thoroughly until completely dissolving and store at 20 C Resuspend lyophillized Carrier RNA by addition of 1 2 ml RNase free Water to the vial and mix thoroughly until completely dissolving Fill 15 ml 99 7 isopropanol molecular biologic grade into the empty bottle Add 20 ml of 99 7 Isopropanol to the bottle Wash Buffer HLT Mix thoroughly and always keep the bottle firmly closed Add 42 ml of 99 8 Ethanol to the bottle Wash Buffer Mix thoroughly and always keep the bottle firmly closed 2 ml RNase free Water to the vial mix thoroughly until completely dissolving and store at 20 C Resuspend lyophillized Carrier RNA by addition of 2 ml RNase free Water to each vial mix thoroughly until completely dissolving Fill 80 ml 99 7 isopropanol molecular biologic grade into the empty bottle Add 70 ml of 99 7 Isopropanol to the bottle Wash Buffer HLT Mix thoroughly and always keep the bottle firmly closed Add 140 ml of 99 8 Ethanol to the bottles Wash Buffer Mix thoroughly and always keep the bottle firmly closed Invisorb Spin Universal Kit Version 0515 Symbo
33. own or by vortexing Transfer the sample into the RTA Spin Filter put into a RTA Receiver Tube Close the cap and centrifuge for 2 minutes at 11 000 x g 11 000 rpm Discard the RTA Receiver Tube with filtrate and place the RTA Spin Filter into a new Receiver Tube 3 First Washing of the RTA Spin Filter Add 600 ul Wash Buffer HLT to the RTA Spin Filter and centrifuges at 11 000 x g 11 000 rpm for 1 min Discard the Receiver Tube with filtrate and place the RTA Spin Filter into a new Receiver Tube 4 Second Washing of the RTA Spin Filter Add 700 ul Wash Buffer to the RTA Spin Filter and centrifuge at 11 000 x g 11 000 rpm for 1 min nucleic acids include genomic DNA bacterial DNA viral DNA and viral RNA 21 Invisorb Spin Universal Kit Version 0515 5 Ethanol removal Discard the RTA Receiver Tube with filtrate and place the RTA Spin Filter into a new RTA Receiver Tube Remove the residual ethanol by final centrifugation for 4 min at maximum speed Discard the RTA Receiver Tube with filtrate 6 Elution of the DNA RNA Place the RTA Spin Filter into a 1 5 ml Elution Tube Add 60 ul of the Elution Buffer M prewarmed to 65 C directly onto the RTA Spin Filter surface Incubate for 3 minutes at RT and centrifuge at 11 000 x g 11 000 rpm for 1 minute 22 Invisorb Spin Universal Kit Version 0515 Troubleshooting Problem Common errors low concentration of extracted DNA RNA degraded or sheared DNA RNA Prob
34. r modified 11 Invisorb Spin Universal Kit Version 0515 Preparation of starting materials 1 Extraction of NA from blood serum plasma cell free body fluids urine liquor transport media This type of sample can be processed directly without any pre preparations Please keep in mind that the first step in the equipment is premixing of samples Samples have to be at least pipetable mean the presence of clumps and other solid materials leads to clots and prevents a normal workflow of the process We recommend strictly controlling samples for coagulation by mixing several times overhead before usage on the instrument For blood take care that it is well stored and stabilized with EDTA or Citrate Please use Protocol 1 blood for all other materials use Protocol 2 2 Extraction of NA from rinsed liquid from swab samples a the sample will also be used for cultivation Cut off the relevant part of the swab and transfer it into an RNase DNAse free 2 ml tube Add 400 ul physiological saline solutions to the swab and vortex intensely for 2 3 min and incubate for 10 min at RT Take an aliquot for cultivation Transfer 200 ul of the rinsed liquid into a 2 0 ml Safe Lock Tube and follow step 1 for NA extraction from bacteria or follow step 2 for NA extractions from virus in protocol 2 optional If bacterial DNA is processed 20 ul Lysozyme can be added to 200 ul sample follow the instructions of protocol 2 Note This does not inclu
35. rsal Kit Version 0515 General notes on handling RNA RNA is far less stable than DNA It is very sensitive to degradation by endogenous RNases in the biological material and exogenous RNases which are permanently present everywhere in the lab To achieve satisfactory qualitative and quantitative results in RNA preparations contaminations with exogenous RNases has to be reduced as much as possible Avoid handling bacterial cultures cell cultures or other biological sources of RNases in the same lab where the RNA purification is to be carried out All glassware should be treated before use to ensure that it is RNase free Glassware should be cleaned with detergent thoroughly rinsed and oven baked at 240 C for four or more hours before use Autoclaving alone will not completely inactivate many RNases Oven baking will both inactivate RNases and ensure that no other nucleic acids such as Plasmid DNA are present on the surface of the glassware You can also clean glassware with 0 1 DEPC diethyl pyrocarbonate The glassware must stand 12 hours at 37 C and then be autoclaved or heated to 100 C for 15 minutes to remove residual DEPC o Electrophoresis tanks should be cleaned with detergent solution e g 0 5 SDS thoroughly rinsed with RNase free water and then rinsed with ethanol and allowed to dry o Non disposable plasticware should be treated before use to ensure that it is RNase free plastic ware should be thoroughly rinsed with 0 1 M
36. sample preparation 1 carefully apply the sample or solution to the RTA Spin Filter pipet the sample onto the filter without wetting the rim of the column 2 always change pipet tips between liquid transfers we recommend the use of aerosol barrier pipet tips 3 avoid touching the RTA Spin Filter membrane with the pipet tip 9 Invisorb Spin Universal Kit Version 0515 Yield and quality of genomic DNA RNA from Blood The amount of purified DNA RNA in the Invisorb Spin Universal Kit procedure from whole blood depends on the leucocytes content the sample source transport storage and age The typical yield usually expected from the Invisorb Spin Universal Kit is about 1 ug DNA Please keep in mind that a small amount of CarrierRNA in the eluate will elevate the real genomic DNA content Yield and quality of pathogen DNA RNA Different amplification systems vary in efficiency depending on the total amount of nucleic acid present in the reaction Eluates derived by this kit will contain Carrier RNA which will greatly exceed the amount of the isolated NA Yields of viral nucleic acids isolated from biological samples are usually low concentrated and therefore almost impossible to determine photometrically gt Keep in mind that the Carrier RNA 5 ug per 200 ul sample will account for most of the present NA The kit is suitable for downstream analysis with NAT techniques for examples qPCR RT qPCR LAMP LCR Diagnostic a
37. ssays should be performed according to the manufacturer s instructions Quantitative RT PCR is recommended for determination of viral RNA yield In Gel Electrophoresis and in Capillary Electrophoresis DNA extracted with the provided kit looks like degraded cause the kit contains Carrier RNA this is poly A RNA in fragments of 100 up to 1000 bases The kit is not dedicated for applications using this kind of analysis Sampling storage and preparing of starting materials Please read the instructions carefully and conduct the prepared procedure Sampling and storage For reproducible and high yields appropriate sample storage is essential Yields may vary from sample to sample depending on factors such as health of the donor sample age kind of sample transport and storage conditions Blood Best results are obtained using fresh blood samples Blood samples stabilized with EDTA or citrate but not heparin can be stored at room temperature 18 25 C for 2 3 hours For short time storage up to 24h samples should be stored at 4 8 C For long term storage we recommend to freeze the samples at 20 C or 80 C Avoid multiple thawing and freezing cycles of the sample s before isolating the DNA RNA Serum and plasma After collection and centrifugation serum plasma from blood treated with anticoagulants like EDTA or citrate but not with heparin synovial fluid samples or other cell free body fluids swabs as well as stool samples ca
38. st be pelleted while the supernatant is completely removed urea contaminations can inhibit PCR reactions Best results are obtained with fresh peletted material or bacteria pellets that has been immediately frozen and stored at 20 C or 80 C Repeated freezing and thawing of stored samples should be avoided since this leads to reduced DNA size The amount of purified DNA from max 15 50 ml urine depends on the included bacteria titre Stool samples Best results are obtained with fresh material Stool samples contain DNases and RNases which realize quickly DNA and RNA digestion and degradation The sample may be stored at 80 C Cell culture supernatants Best results are obtained with fresh material or material that has been immediately frozen and stored at 20 C or 80 C after winning of the cell culture supernatant Repeated freezing and thawing of stored samples can influence the sensitivity Amniotic fluid The protocol works with fresh amniotic fluid and amniotic fluid stored at 4 C Best results are obtained using freshly collected samples The sample can be stored at 4 C for at least 3 month For long term storage the samples can be stored at 20 C Avoid multiple thawing and freezing cycles of the sample s before isolating the DNA RNA 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 o
39. t disturbing the bacterial pellet Resuspend the bacterial pellet in 200 ul distilled water or RNAse free water and transfer the sample into a 2 0 ml Safe Lock Tube and follow step 1 for NA extraction from bacteria or follow step 2 in protocol 2 For viral NA use the origin sample and follow step 2 in protocol 2 optional If bacterial DNA is processed 20 ul Lysozyme can be added to 200 ul sample follow the instructions of protocol 2 12 Invisorb Spin Universal Kit Version 0515 Viscous sample Transfer e g 1 ml of tracheal secretes or BAL into a RNase and DNAse free tube and add 1 ml NAC Buffer order number 1033221100 ratio sample to buffer must be 1 1 or 2 ml saturated Acetylcysteine ACC solution to the sample ratio sample to buffer must be 1 2 Incubate the mixture for 10 min at 95 C to reduce the viscosity and centrifuge at 11 100 x g 11 000 rpm for 3 min For isolation of viral NA take a 200 ul aliquote into a 2 0 ml Safe Lock Tube and follow step 2 in protocol 2 For isolation of bacterial DNA discard the supernatant without disturbing the bacterial pellet directly Resuspend the bacterial pellet in 200 ul distilled water or RNase free water and transfer it into a 2 0 ml Safe Lock Tube and follow step 1 in protocol 2 5 Extraction of viral NA from supernatant of stool suspension Transfer 100 u 100 mgl stool sample into a 2 ml tube and add 900 ul RNase free Water Vortex the sample for 30 s followed by a 1 min centrifugation
40. ular 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 Invisorb Spin Universal 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 Invisorb Spin Universal 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 2903 or contact your local distributor 4 Invisorb Spin Universal Kit Version 0515 Intended use The Invisorb Spin Universal Kit is designed for extraction and purification of DNA RNA from 200 ul sample material for blood sample only 100 ul using the RTA Spin Filter system with capped spin columns The nucleic acid isolation protocol is suitable for routinely preparation of DNA RNA from fresh or frozen samples For reproducible and high
41. where the EU Directive 98 79 EC on in vitro medical devices is not recognized Trademarks Invisorb 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 is a registered trademark 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 Contents Kit contents of the Invisorb Spin Universal Kit 3 Symbols 4 Storage 4 Quality Control 4 Intended use 5 Product use limitation 5 Safety information 6 Product characteristics of the Invisorb Spin Universal Kit 7 Principle and procedure 8 Yield and quality of genomic DNA RNA from blood 8 Yield and quality of viral DNA RNA 8 Important notes 9 Equipment and reagents to be supplied by user 9 Important indications 10 Sampling storage and preparing of starting materials 10 Preparation of starting materials 12 Lysis procedures 14 Preparation of a master mix 14 Extraction control 14 Instructions 15 Scheme for the Isolation of genomic DNA from blood 16 Protocol 1 Isolation of genomic DNA from blood 17 Scheme for the simultaneous isolation of pathogen DNA
42. yields an appropriate sample storage is essential see Sampling and storage of the starting material page 8 The Invisorb Spin Universal Kit is the ideal tool for reliable and fast simultaneous isolation of high quality genomic bacterial and viral DNA as well as viral RNA from fresh or frozen human or mammalian blood serum plasma swabs cerebrospinal fluid cell culture supernatants urine and other cell free body fluids The kit can furthermore be used for the isolation of high quality genomic bacterial and viral DNA as well as viral RNA from the same kind of samples but coming from animals The amount of blood depends on the kind of animals Blood samples have to be stabilized with EDTA or citrate not heparin For reproducible high yields an appropriate sample storage and quick operation under the rules for RNA and DNA operation is essential The purified DNA and or RNA is ready to use for subsequent downstream analysis The isolation protocols as well as all buffers are optimized to provide high yield and purity of the extracted nucleic acids The procedure requires minimal interaction by the user allowing safe handling of potentially infectious samples 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 findings THE PRODUCT IS INTENDED FOR USE BY PROFESSIONALS ONLY SUCH AS TECHNIC
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