Invisorb Spin Blood Mini Kit User manual
Contents
1. genomic DNA Please read protocols prior the start of the preparation carefully Transfer max 200 ul of the blood into a 1 5 ml Receiver Tube Add 200 ul Lysis Buffer HL and mix 5 times by pipetting up and down Incubate for 3 min at 56 C while continuously shaking Add 20 ul Proteinase K and vortex shortly Incubate for 5 min at 56 C while continuously shaking Add 200 ul Binding Buffer HL follow preparing instructions and mix by pipetting up and down four times or vortexing Take a RTA Spin Filter Set Transfer lysate onto RTA Spin Filter Centrifuge for 2 min at 11 000 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 500 ul Pre Wash Buffer Centrifuge for 1 min at 11 000 x g 11 000 rpm Discard the filtrate and the RTA Receiver Tube Place RTA Spin Filter to a new 2 0 ml RTA Receiver Tube Add 700 ul Wash Buffer Centrifuge for 1 min at 11 000 x g 11 000 rpm Discard the filtrate and the RTA Receiver Tube repeat the step but add the RTA Spin Filter to the same RTA Receiver Tube then centrifuge for 4 min at maximum speed for ethanol removal Place the RTA Spin Filter into a 1 5 ml Receiver Tube Add 100 200 ul of Elution Buffer preheated to 56 C Incubate for 1 min at room temperature Centrifuge for 1 min at 11 000 x g 11 000 rpm Discard RTA Spin Filter Close the 1 5 ml Receiver Tube and store the DNA sample at 4 C 10 Invisorb Spin2. Blood Mini Kit 0213 Protocol 1 DNA Isolation from 1 200 yl human and mammalian whole blood or 1 30 ul buffy coat Please read the instructions carefully and conduct the prepared procedure Attention Please be aware that you have to prepare the Binding Buffer HL see instruction page 9 Important Transfer the needed amount of Elution Buffer into 2 0 ml Receiver Tube not included in the kit and place the tube at 56 C 1 Transfer 1 200 ul whole blood or 1 30 ul buffy coat into a 1 5 ml reaction tube If sample volume is lower than 200 ul equilibrate with 1 x PBS Buffer or distilled water to 200 ul 2 Add 200 ul Lysis Buffer HL mix by pipetting up and down 5 times and incubate for 3 min at 56 C while continuously shaking Add 20 ul Proteinase K and vortex shortly 3 Incubate the reaction tube for 5 min at 56 C while continuously shaking on a thermomixer Note If you should use a water bath please vortex the sample during lysis 2 5 times 4 Add 200 ul Binding Buffer HL and mix the sample by vortexing or pipetting up and down for 4 5 times Take a RTA Spin Filter Set Transfer the mixture into the RTA Spin Filter Close the RTA Spin Filter and incubate for 1 min 5 Centrifuge for 2 min at 11 000 x g 11 000 rpm Discard the filtrate and place the RTA Spin Filter in a new 2 0 ml RTA Receiver Tube 6 Add 500 ul Pre Wash Buffer to the RTA Spin Filter Close the RTA Spin Filter Centrifuge for 1 min at 11 000
3. may lead to inoperability therefore neither a warranty nor guarantee in this case will be given neither implied nor express The user is responsible to validate the performance of the STRATEC Molecular Product for any particular use STRATEC Molecular does not provide for validation of performance characteristics of the Product with respect to specific applications STRATEC Molecular Products may be used e g in clinical diagnostic laboratory systems conditioned upon the complete diagnostic system of the laboratory the laboratory has been validated pursuant to CLIA 88 regulations in the U S or equivalents in other countries All Products sold by STRATEC Molecular are subject to extensive quality control procedures according to ISO 9001 2000 and ISO EN 13485 and are warranted to perform as described herein Any problems incidents or defects shall be reported to STRATEC Molecular immediately upon detection thereof The chemicals and the plastic parts are for laboratory use only they must be stored in the laboratory and must not be used for purposes other than intended The Product with its contents is unfit for consumption 4 Invisorb Spin Blood Mini Kit 0213 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 appropri
4. x g 11 000 rpm Discard the filtrate and place the RTA Spin Filter in a new RTA Receiver Tube 7 Add 700 ul Wash Buffer and centrifuge for 1 min at 13 000 x g 12 000 rpm Discard the filtrate and place the RTA Spin Filter in a new RTA Receiver Tube 8 Add 700 ul Wash Buffer and centrifuge for 1 min at 11 000 x g 11 000 rpm Discard the filtrate 9 Place the RTA Spin Filter again into the 2 0 ml RTA Receiver Tube Centrifuge for 4 min at maximum speed to eliminate the ethanol completely 10 Place the RTA Spin Filter in a new 1 5 ml Receiver Tube Add 200 ul of the preheated 56 C Elution Buffer Incubate at room temperature for 1 min 11 Centrifuge at 11 000 x g 11 000 rpm for 1 min Discard the RTA Spin Filter Note The DNA can also be eluted with a lower volume of Elution Buffer depends on the expected yield of genomic DNA But pay attention that the minimum volume for the elution is 30 ul and that this volume can reduce the maximum yield If quite large amount of DNA is expected the volume of Elution Buffer can be increased Note The centrifugation steps were made with the Centrifuge 5415 D from Eppendorf The indicated rpm amounts are referring to this centrifuge 11 Invisorb Spin Blood Mini Kit 0213 Protocol 2 DNA Isolation from non mammalian blood sample material If you want to use bird e g chicken or fish blood that contain nucleated erythrocytes the use of only 10 15 ul of starting material is recomme
5. H20 96 100 ethanol 1 x PBS optional O O O O 0 06 0 O Oo O The Invisorb Spin Blood Mini 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 2 Propanol Applichem Sigma Rotipuran gt 99 7 p a ACS ISO 2 Propanol fur die Molekularbiologie 2 Propanol Order no 6752 Order no A3928 Order no 59304 1L F Important indications 1 Process only as much blood samples as the microcentrifuge allows to process 2 Blood sample and buffers should be thoroughly mixed and should have room temperature 3 The elution can be done by using lower amount of Elution Buffer This may result in a higher concentration of DNA But pay attention that minimum volume for elution is 30 ul but this will reduce the yield Elution volume between 2 x 30 ul up to 200 ul will realize comparable results 4 The eluted DNA volume can be lower than the added Elution Buffer volume Elution Buffer should be preheated to 56 C 5 The Elution Buffer doesn t contain EDTA 6 The yield can be increased if the incubation time with preheated Elution Buffer will be prolonged 7 Old blood samples often contains coagulates if coagulates or cryoprecipitate formed during thawing of frozen samples are visible avoid aspirating them they could clog the Spin Filter membrane 9 Invisorb Spin Blood Mini Kit 0213 Scheme of the Invisorb Spin Blood Mini Kit
6. Receiver Tube not included in the kit and place the tube at 56 C Preparation of the starting material Fresh material o 1 20 ul bone marrow Dried material for example on hematological slides o Moisten the dried material with a drop of PBS o Add 180 ul PBS to a 1 5 ml Receiver Tube not provided o Scrape cytological material into the Receiver Tube using the edge of a clean slide o Dissolve the resulting sludge by pipetting up and down Sample Lysis 1 Transfer the starting material into a 1 5 ml reaction tube Equilibrate with 1 x PBS Buffer e g to 200 ul 2 Add 200 ul Lysis Buffer HL mix by pipetting up and down 5 times and incubate for 3 min at 56 C while continuously shaking Add 20 ul Proteinase K and vortex shortly Important Vortex the sample for 10 sec An incomplete mixing will reduce quality and yield of the isolated DNA 3 Incubate the reaction tube for 5 min at 56 C while continuously shaking on a thermomixer Note If you should use a water bath please vortex the sample during lysis 2 5 times Proceed as described in protocol 1 steps 4 11 13 Invisorb Spin Blood Mini Kit 0213 Troubleshooting Problem Cause Comments and suggestions low amount insufficient cell lysis increase lysis time with Lysis Buffer HL of DNA reduce amount of starting material continuously shaking improves lysis efficiency insufficient cell lysis due to repeat the DNA purification procedure wit
7. ate 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 liquid waste generated by the Invisorb Spin Blood Mini procedures for residual infectious materials Contamination of the liquid waste with residual infectious materials is highly unlikely but cannot be excluded completely Therefore liquid waste must be considered infectious and be handled and discarded according to local safety regulations European Community risk and safety phrases for the components of the Invisorb Spin Blood Mini Kit to which they apply are listed below as follows Lysis Buffer HL Proteinase K waming danger H315 319 334 335 P280 305 351 310 405 H225 319 336 P210 233 305 351 338 H315 Causes skin irritation H319 Causes serious eye irritation H225 Highly flammable liquid and vapour H319 Causes serious eye irritation H336 May cause drowsiness or dizziness 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 if
8. e clear plasma is buffy coat containing concentrated leukocytes that can be easily distinguished from the erythrocytes in the bottom layer An enrichment factor of 10 is expected from such a procedure Due to the enriched leukocyte content be aware to avoid overloading the DNA purification procedure Bone Marrow Best results are obtained with fresh material It can be stored for 2 3 h at 4 C for longer storage freeze the sample at 20 C But often the sample will be dried The have to be stored cooled at 4 C in a dry surrounding Procedure Lysis Samples are lysed at elevated temperatures Lysis is performed in the presence of Lysis Buffer HL and Proteinase K Binding genomic DNA By adding Binding Buffer HL to the lysate optimal binding conditions will be adjusted Each lysate is then applied to an RTA Spin Filter and genomic DNA is adsorbed to the membrane Removing residual contaminations Contaminants are efficiently washed away using Pre Wash Buffer and Wash Buffer while the genomic DNA remains bound to the membrane Elution Genomic DNA is eluted from the RTA Spin Filter using 30 200 ul Elution Buffer The eluted DNA is ready for use in different downstream applications Eluted DNA stored at 4 8 C is stable for 2 months for more than 5 years if stored at 20 C 7 Invisorb Spin Blood Mini Kit 0213 Yield and quality of genomic DNA The amount of purified DNA using the Invisorb Spin Blood Mini Kit procedure depe
9. elution The purification procedure is rapid and requires neither phenol chloroform extraction nor alcohol precipitation and requires minimal interaction by the user allowing safe handling of potentially infectious samples The procedure is designed to avoid sample to sample cross contamination Due to the high purity the isolated genomic DNA is ready to use for a broad panel of downstream applications see below or can be stored at 20 C for subsequent use Downstream Application o PCR o Restriction Enzyme Digestion o SNP Analysis o HLA typing o Cloning To purify genomic DNA in 96 format STRATEC Molecular offers the Invisorb Blood Mini HTS 96 Kit for use in a centrifuge and on common laboratory automated workstations Furthermore STRATEC Molecular offers the InviMag Blood Mini Kits for DNA isolation using magnetic beads To purify genomic DNA from large volumes of blood STRATEC Molecular offers the Invisorb Spin Blood Midi Kit max 2 ml the Invisorb Spin Blood Maxi Kit max 10 ml the Invisorb Blood Universal Kit 1 10 ml For blood stains STRATEC Molecular offers the Invisorb Spin Forensic Kit For further information please contact Tel 49 0 30 9489 2901 2910 in Germany and from foreign countries Tel 49 0 30 9489 2907 or your local distributor The PCR process is covered by US Patents 4 683 195 and 4 683 202 and foreign equivalents owned by Hoffmann La Roche AG 6 Invisorb Spi
10. er than 5 x 10 200 ul problems with subsequent applications e g in PCR ethanol in the eluted DNA salt in the eluate reduced sensitivity of amplification reaction verify if the recommended centrifugation time was reached increase centrifugation time for the elimination of ethanol if necessary Wash Buffer should be stored at and used at RT verify Wash Buffer on the precipitation of salt If there are precipitations dissolve this by careful warming up to 30 C adjust the volume of eluate added as template in the amplification reaction A260 A280 ratio for purified DNA is high high level of residual RNA in future DNA preparations prepare an RNase digestion step add 10 ul RNase A 10 mg ml to the eluted DNA vortex shortly and incubate 10 min at RT A260 A280 ratio for purified DNA is low insufficient cell lysis and protein degradation due to decreased Proteinase K activity insufficient lysis due to insufficient mixing with Lysis Buffer HL see above under low amount of DNA see above under low amount of DNA 15 Invisorb Spin Blood Mini Kit 0213 Appendix General notes on handling DNA Starting material This kit is designed for extraction of DNA from blood but even human blood is different between individuals depending on age health and conditions of life If you are using blood from animals keep in mind that lyses conditions of blood differ depending on the
11. filtrate and place the RTA Spin Filter in a new RTA Receiver Tube 8 Add 700 ul Wash Buffer and centrifuge for 1 min at 11 000 x g 11 000 rpm Discard the filtrate 9 Place the RTA Spin Filter again into the 2 0 ml RTA Receiver Tube Centrifuge for 4 min at maximum speed to eliminate the ethanol completely 10 Place the RTA Spin Filter in a new 1 5 ml Receiver Tube Add 200 ul of the preheated 56 C Elution Buffer Incubate at room temperature for 1 min 11 Centrifuge at 11 000 x g 11 000 rpm for 1 min Discard the RTA Spin Filter Note The DNA can also be eluted with a lower volume of Elution Buffer depends on the expected yield of genomic DNA But pay attention that the minimum volume for the elution is 30 ul and that this volume can reduce the maximum yield If quite large amount of DNA is expected the volume of Elution Buffer can be increased Note The centrifugation steps were made with the Centrifuge 5415 D from Eppendorf The indicated rpm amounts are referring to this centrifuge 12 Invisorb Spin Blood Mini Kit 0213 Protocol 3 DNA isolation from CFS and bone marrow For the isolation and purification of DNA from small amounts of various human and mammalian samples Please read the instructions carefully and conduct the prepared procedure Attention Please be aware that you have to prepare the Binding Buffer HL see instruction page 9 Important Transfer the needed amount of Elution Buffer into an 2 0 ml
12. formation see page 7 Do not use damaged kit components since their use may lead to poor kit performance o Always change pipet tips between liquid transfers To avoid cross contamination we recommend the use of aerosol barrier pipet tips o All centrifugation steps should be carried out at room temperature 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 until the samples are lysed o This kit should only be used by trained personnel O O O O Preparing reagents and buffers Adjust the thermomixer to 56 C Warm up the needed amount of Elution Buffer to 56 C 100 200 ul Elution Buffer are needed per sample Label the needed amount of RTA Spin Filter lid Label the needed amount of 1 5 ml Receiver Tubes per sample 1 Receiver Tube Add the needed ul ddH20 to reaction tube with Proteinase K see below Vortex for 5 s Add the needed amount of ethanol to the Pre Wash Buffer and Wash Buffer Na Sole 5 DNA extractions add 250 ul dd H20 to Proteinase K mix thoroughly vortex 5s and store at 20 C Binding Buffer HL pre Wash Buffer and Wash Buffer a
13. h a new sample anq decreased Proteinase K freshly prepared Proteinase K stock solution be sure to activity store the stock solution at 20 C ensure that Proteinase K is not added directly to Lysis Buffer HL insufficient lysis due to repeat the DNA purification procedure with a new sample be insufficient mixing with sure to mix the sample and Lysis Buffer HL immediately anq Lysis Buffer HL thoroughly by pipetting up and down 5 times or by pulse vortexing inefficient binding of DNA to overloading RTA Spin Filter reduces yield the membrane use correct amount of Binding Buffer HL mix sample with Binding Buffer HL by pipetting up and down 4 5 times or by vortexing 5 sec prior to transfer the sample onto the Spin Filter low percentage alcohol used repeat purification procedure with a new sample instead of 96 100 incomplete elution increase incubation time with preheated Elution Buffer to 5 10 min elute twice with each 100 ul Elution Buffer use higher volume of Elution Buffer low DNA concentration in the elute the DNA with lower volume of Elution Buffer sample pH of water incorrect acidic low pH may reduce DNA yield Ensure that the pH of the water is at least 7 0 or use Elution Buffer contains only 10 mM Tris HCL no EDTA colored insufficient cell lysis see above residues remain on the no Binding Buffer HL added repeat the purification procedure with a new sample RTA Spin filter to the lysate before loading after wa
14. iMag Blood DNA Mini Kit KF96 5 x 96 preparations 7431300200 Possible suppliers for lsopropanol Carl Roth 2 Propanol Applichem Sigma Rotipuran gt 99 7 p a ACS ISO Order no 6752 Order no A3928 17 2 Propanol fur die Molekularbiologie 2 Propanol Order no 59304 1L F Invisorb Spin Blood Mini Kit 0213 Stratecee molecular STRATEC Molecular GmbH Robert Rossle Str 10 13125 Berlin Germany Phone 49 30 94 89 29 01 Fax 49 30 94 89 29 09 E mail info berlin stratec com www stratec com 1A3a01 02 2013
15. keep the bottle firmly closed Add 105 ml of 96 100 ethanol to Wash Buffer mix thoroughly and always keep the bottle firmly closed Invisorb Spin Blood Mini Kit 0213 Symbols Manufacturer rm j Lot number Catalogue number Date of manufacture Expiry date Consult operating instructions Temperature limitation 9x BLE F Do not reuse Storage All buffers and kit components of the Invisorb Spin Blood Mini Kit except dissolved Proteinase K should be stored at room temperature RT and are stable for 12 months under these conditions Room temperature RT is defined as range from 15 30 C Dissolved Proteinase K stored at 20 C is stable for 12 months but repeated freezing and thawing should be avoided Aliquotation and storage at 20 C is recommended Pre Wash Buffer and Wash Buffer charged with ethanol should be appropriately sealed and stored at room temperature If there are any precipitates within the provided solutions dissolve these precipitates by carefully warming up to room temperature up to 30 C Quality control and product warranty STRATEC Molecular warrants the correct function of the Invisorb Spin Blood Mini 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 Molecular will check the lot and if STRATEC Molecu
16. lar 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 ISO 9001 2000 and ISO EN 13485 certified Quality Management System the performance of all components of the Invisorb Spin Blood Mini 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 Blood Mini Kit or other STRATEC Molecular products please do not hesitate to contact us A copy of STRATEC Molecular s terms and conditions can be obtained upon request or are presented at the STRATEC Molecular webpage For technical support or further information please contact from Germany 49 0 30 9489 2901 2910 from abroad 49 0 30 9489 2907 or contact your local distributor 3 Invisorb Spin Blood Mini Kit 0213 Intended use The Invisorb Spin Blood Mini Kit is the ideal tool for a fast and convenient manual isolation and purification of genomic DNA from max 200 ul fresh frozen or old human and from max 100 ul mammalian blood as well from buffy coat max 30 ul or non mammalian blood max 25 ul For reproducible and high yields appropriate sample storage is essential The purified DNA can be used for in vitro diagnostic anal
17. n Blood Mini Kit 0213 Principle and procedure The Invisorb Spin Blood Mini Kit procedure comprises following steps 1 lysis of sample material 2 binding the genomic DNA to the membrane of RTA Spin Filter 3 washing the membrane and elimination of ethanol 4 elution of genomic DNA This manual contains 3 protocols according to the different requirements of the starting materials Sampling and storage of starting material Blood and Buffy Coat Mammalian blood samples stabilized with EDTA or Citrate can be stored at room temperature for 2 3 hours for short time storage up to 24 h samples may be stored at 4 C For long term storage we recommend freezing samples at 20 C or 80 C Multiple thawing and freezing before isolating the DNA should be avoided If cryoprecipitate formed during thawing of frozen samples are visible avoid aspirating them they could clog the RTA Spin Filter membrane Various different primary tubes blood collection system e g Sarstedt Greiner and anticoagulants except heparin can be used to collect blood samples for the Invisorb procedure Buffy coat is a whole blood fraction of enriched leukocyte cells To prepare and extract a buffy coat layer the following procedure is recommended The use of a whole blood sample anticoagulants EDTA citrate not heparin with a sedimented cellular fraction from staying overnight at 4 C is recommended The resulting bright mid section overlaid by th
18. nded Please read the instructions carefully and conduct the prepared procedure Attention Please be aware that you have to prepare the Binding Buffer HL see instruction page 9 Important Transfer the needed amount of Elution Buffer into a 2 0 ml Receiver Tube not included in the kit and place the tube at 56 C 1 Transfer 1 25 ul whole blood into a 1 5 ml reaction tube Equilibrate with 1 x PBS Buffer e g to 200 ul 2 Add 200 ul Lysis Buffer HL mix by pipetting up and down 5 times and incubate for 3 min at 56 C while continuously shaking Add 20 ul Proteinase K and vortex shortly 3 Incubate the reaction tube for 5 min at 56 C while continuously shaking on a thermomixer Note If you should use a water bath please vortex the sample during lysis 2 5 times 4 Add 200 ul Binding Buffer HL and mix the sample by vortexing or pipetting up and down for 4 5 times Take a RTA Spin Filter Set Transfer the mixture into the RTA Spin Filter Close the RTA Spin Filter and incubate for 1 min 5 Centrifuge for 2 min at 11 000 x g 11 000 rpm Discard the filtrate and place the RTA Spin Filter in a new 2 0 ml RTA Receiver Tube 6 Add 500 ul Pre Wash Buffer to the RTA Spin Filter Close the RTA Spin Filter Centrifuge for 1 min at 11 000 x g 11 000 rpm Discard the filtrate and place the RTA Spin Filter in a new RTA Receiver Tube 7 Add 700 ul Wash Buffer and centrifuge for 1 min at 11 000 x g 11 000 rpm Discard the
19. nds on the sample type and the number of cells in the sample depending from the patient s age and health situation sample source transport conditions storage and age of the sample Typically a 200 ul sample of whole blood cells from a healthy individual will yield 3 10 ug of DNA If higher yields are required use Invisorb Spin Blood Midi Kit or Invisorb Spin Blood Maxi Kits with up to 2 ml or up to 10 ml blood respectively Samples with elevated white blood cell WBC counts ranging from 3 x 10 to 1 x 10 cells ml give a higher yield For most whole blood samples a single elution with 200 ul Elution Buffer is sufficient For samples with elevated white blood cell approximately 80 of the DNA will elute in the first 200 ul and up to 20 more in the next 200 ul Yield and quality of isolated genomic DNA is suitable for any molecular diagnostic detection system The diagnostic tests should be performed according to manufacturers specifications Important points before starting a protocol Immediately upon receipt of the Product inspect the Product and its components as well as the package for any apparent damages correct quantities and quality If there are any unconformities you have to notify STRATEC Molecular in writing with immediate effect upon inspection thereof If buffer bottles are damaged contact the STRATEC Molecular Technical Services or your local distributor In case of liquid spillage refer to Safety In
20. 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 2013 STRATEC Molecular all rights reserved Contents Kit contents of the Invisorb Spin Blood Mini Kit Symbols Storage Quality control Intended use Product use limitation Safety information Product characteristic of the Invisorb Spin Blood Mini Kit Principle and procedure Sampling and storage of starting material Yield and quality of genomic DNA Important points before starting a protocol Preparing reagents and buffers Reagents and equipment to be supplied by user Important indications Scheme Protocol 1 DNA Isolation from 1 200 ul human and mammalian whole blood or 1 30 ul buffy coat Protocol 2 DNA Isolation from non mammalian blood sample material Protocol 3 DNA isolation from bone marrow Troubleshooting Appendix Ordering information O O O ON NO O FF A WO WO W ND 12 13 14 16 17 1 Invisorb Spin Blood Mini Kit 0213 Kit contents of the Invisorb Spin Blood Mini Kit Store dissolved Proteinase K at 20 C Store all other kit component
21. present and easy to do Continue rinsing P210 Keep away from heat sparks open flames hot surfaces No smoking P233 Keep container tightly closed P310 Immediately calla 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 in USA 1 800 535 5053 5 Invisorb Spin Blood Mini Kit 0213 Product characteristic of the Invisorb Spin Blood Mini Kit Time for Starting material Yield preparation Ratio 1 200 ul fresh frozen or old up to 10 ug in average approx A260 A 280 human or other mammalian about 6 ug depends on 25 min 1 7 2 0 whole blood EDTA citrate 1 30 ul buffy coat 1 25 ul fresh frozen or old non mammalian blood 1 20 ul bone marrow amount of lymphocytes sample source sample transport sample storage and age of the sample The Invisorb Spin Blood Mini Kit provides a very efficient procedure for isolation of high quality DNA directly from fresh frozen or old blood samples treated with citrate or EDTA or buffy coat samples The kit is designed for simultaneous processing of multiple samples Prior separation of leukocytes is not necessary The whole blood sample is lysed in an optimized lysis buffer and proteins are degraded during the lysis with Proteinase K The DNA binds to filter membrane followed by washing steps and the final
22. re ready to use 8 Invisorb Spin Blood Mini Kit 0213 50 DNA extractions add 12 ml 99 7 Isopropanol to the Binding Buffer HL Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times add 1 1 ml dd H20 to Proteinase K mix thoroughly vortex 5s and store at 20 C add 30 ml of 96 100 ethanol to Pre Wash Buffer add 42 ml of 96 100 ethanol to Wash Buffer mix thoroughly and always keep the bottle firmly closed 250 DNA extractions add 24 ml 99 7 Isopropanol to each Binding Buffer HL Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times add 1 1 ml dd H20 to Proteinase K mix thoroughly vortex 5s and store at 20 C add 80 ml of 96 100 ethanol to Pre Wash Buffer add 105 ml of 96 100 ethanol to Wash Buffer mix thoroughly and always keep the bottle firmly close Reagents and equipment 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 These are available online in convenient and compact PDF format at www stratec com under each STRATEC Molecular kit and kit component Microcentrifuge Thermomixer for 56 C Measuring cylinder 250 ml Disposable gloves Pipette and pipette tips Vortexer lsopropanol Reaction tubes 1 5 ml or 2 0 ml dd
23. s at room temperature RT 5 DNA extractions 50 DNA extractions 250 DNA extractions Catalogue No 1031100100 1031100200 1031100300 Lysis Buffer HL 2ml 15 ml 60 ml 2x1ml 4ml 2x8ml Binding Buffer HL ready to use final volume 16 ml final volume 2 x 32 ml Elution Buffer 2ml 15 ml 60 ml Proteinase pele Aan oe ae Pre KWash Bunter je final nie 60 ml final Sinaia ml WASA Butar E final a os ml final ee ag ml RTA Spin Filter Set 5 50 5 x 50 RTA Receiver Tubes 15 3 x 50 15 x 50 1 5 ml Receiver Tubes 10 2x 50 10 x 50 Manuals 1 1 1 Initial steps Add 250 ul Add 12 ml 99 7 Add 24 ml 99 7 ddH 0 to Proteinase K mix thoroughly and store at 20 C Isopropanol to the Binding Buffer HL Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times Add 1 1 ml ddH 0 to Proteinase K mix thoroughly and store at 20 C Add 30 ml of 96 100 ethanol to Pre Wash Buffer mix thoroughly and always keep the bottle firmly closed Add 42 ml of 96 100 ethanol to Wash Buffer mix thoroughly and always keep the bottle firmly closed Isopropanol to each Binding Buffer HL Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times Add 1 1 ml ddH20 to Proteinase K mix thoroughly and store at 20 C Add 80 ml of 96 100 ethanol to Pre Wash Buffer mix thoroughly and always
24. shing onto the RTA Spin Filter inefficient washing wash again with Wash Buffer Pre Wash Buffer and Wash ensure that Pre Wash Buffer and Wash Buffer concentrated Buffer prepared incorrectly were diluted with the correct volume of pure ethanol Repeat the purification with a new sample old material repeated frozen old blood may lead to colored membrane of the RTA Spin material Filter this color will not be eluted during purification yield and quality of DNA is reduced based on sample storag degraded or incorrect storage of ensure the sample is harvested and stored as described on sheared DNA starting material page 8 old material avoid repeated thawing and freezing of the material old material often contains degraded DNA 14 Invisorb Spin Blood Mini Kit 0213 Problem Cause Comments and suggestions clogged incorrect storage of blood stored longer time at RT may form clumps of proteins Spin Filter starting material etc Prevent a transfer of this clumps into the sample insufficient lysis too much starting material cryoprecipitates have formed in blood due to related freezing and thawing do not use blood that has been frozen and thawed more than once prevent a transfer of cryoprecipitateg into the sample perform isolation as described in protocol 2 increase lysis time with Lysis Buffer HL increase centrifugation time and or speed reduce amount of starting material concentration of leukocytes in samples was great
25. species Also remember that non mammalian blood contains erythrocytes with nuclei So for special applications adaptation of starting volumes and lyses time may be recommended Nature of DNA The length and delicate physical 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 Pipe
26. stratecee molecular User manual Invisorb Spin Blood Mini Kit For genomic DNA purification from fresh frozen or old human blood with common anticoagulants EDTA Citrate as well from buffy coat and bone marrow 1031100x00 eaat STRATEC Molecular GmbH D 13125 Berlin Instruction for the Invisorb Spin Blood Mini Kit The Invisorb Spin Blood Mini Kit is the ideal tool using the Invisorb technology for a fast efficient and simple manual isolation and purification of genomic DNA from max 200 ul fresh frozen or old human blood with common anticoagulants EDTA Citrate as well from buffy coat and bone marrow max 30 ul The purified DNA can be used for in vitro diagnostic analysis The kit is also useful for isolation of genomic DNA from max 200 ul of non human mammalian blood or from up to 25 ul non mammalian blood e g birds or fishes The kit is neither validated for the isolation of genomic DNA from tissue serum plasma synovial fluid and urine nor from bacteria stool sample fungi parasites or the purification of total RNA The application of the kit for isolation and purification of viral DNA has not been evaluated Compliance with EU Directive 98 79 EC on in vitro medical devices Not for in vitro diagnostic use in countries where the EU Directive 98 79 EC on in vitro medical devices is not recognized Trademarks Invisorb Registered marks trademarks etc used in this document even when
27. tting 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 DNA Yield The amount of purified DNA from the whole blood depends on the leucocytes content sample source transport storage and age Various different primary tubes and anticoagulants except heparin can be used to collect blood samples for the Invisorb procedure 16 Invisorb Spin Blood Mini Kit 0213 Ordering information Product Package size Catalogue No Invisorb Spin Blood Mini Kit 5 preparations 1031100100 Invisorb Spin Blood Mini Kit 50 preparations 1031100200 Invisorb Spin Blood Mini Kit 250 preparations 1031100300 Lysis Buffer HL 15 ml 1031101100 Binding Buffer HL 15 ml 1031102100 Pre Wash Buffer add 30 ml 30 ml 1031103300 Wash Buffer add 42 ml 18 ml 1031103400 Elution Buffer 15 ml 1031104000 Invisorb Spin Blood Midi Kit 50 preparations 1031110300 Invisorb Spin Blood Maxi Kit 50 preparations 1031120300 Invisorb Blood Universal Kit 500 ml 1031150200 Invisorb Blood Universal Kit 1000 ml 1031150300 Invisorb Blood Mini 96 HTS C 4 x 96 preparations 7031300300 Invisorb Blood Mini 96 HTS C 24 x 96 preparations 7031300400 InviMag Blood DNA Mini Kit KFml 15 preparations 2431110100 InviMag Blood DNA Mini Kit KFml 75 preparations 2431110200 InviMag Blood DNA Mini Kit KF96 1 x 96 preparations 7431300100 Inv
28. ysis only Fresh or frozen whole blood treated with EDTA or citrate but not with heparin from common blood collection systems can be used The protocol for the isolation and all buffers are optimized for a high yield as well as a high purity All hands on steps are reduced to a minimum THE PRODUCT IS INDENTED FOR USE BY PROFESSIONAL USERS ONLY SUCH AS TECHNICIANS PHYSICIANS AND BIOLOGISTS TRAINED IN MOLECULAR BIOLOGICAL TECHNIQUES It is designed to be used with any downstream application employing enzymatic amplification or other enzymatic modifications of DNA followed by signal detection or amplification Any diagnostic results generated by using the sample preparation procedure in conjunction with any downstream diagnostic assay should be interpreted with regard to other clinical or laboratory findings To minimize irregularities in diagnostic results adequate controls for downstream applications should be used The kit is in compliance with EU Directive 98 79 EC on in vitro medical devices But it is not for in vitro diagnostic use in countries where the EU Directive 98 79 EC on in vitro medical devices is not recognized Product use limitation The kit is neither validated for the isolation of genomic DNA from tissue serum plasma synovial fluid and urine nor from bacteria stool sample fungi parasites or the purification of total RNA The included chemicals are only useable once Differing of starting material or flow trace
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