Instruction for the Invisorb Spin Cell Mini Kit

Instruction for the Invisorb ® Spin Cell Mini Kitfor extrations of genomic DNA from cell culture, swabs, sera, plasmaThe Invisorb ® Spin Cell Mini Kit is the ideal tool, using the Invisorb ® technology for a fast, efficientand simple manual isolation and purification of genomic DNA from cultured cells (24 or 96 wellplates), serum, human and animal lavage, fresh or frozen plasma, bone marrow and urine. Thepurified DNA can be used for in-vitro diagnostic analysis.The kit is neither validated for the isolation of cells from tissue, blood, or stool sample, nor frombacteria, fungi, parasites or the purification of total RNA.The application of the kit for isolation and purification of viral DNA has not been evaluated.Trademarks: : Invisorb ® , Invitek. Registered marks, trademarks, etc. used in this document, even when not specificallymarked 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, EP0880535, WO 9728171, WO 9534569, EP 0765335, DE 19506887, DE 10041825.2, WO 0034463.Invisorb ® is a registered trademark of Invitek GmbH.The PCR process is covered by US Patents 4,683,195, and 4,683,202 and foreign equivalents owned by Hoffmann-LaRoche AG.© 2009 Invitek, all rights reserved.1Invisorb ® Spin Cell Mini Kit 032010

ContentsKit contents of the Invisorb ® Spin Cell Mini Kit ....................................................................... 3Symbols ...................................................................................................................................... 4Storage........................................................................................................................................ 4Quality control............................................................................................................................ 4Intended use ............................................................................................................................... 5Product use limitation................................................................................................................ 5Safety information...................................................................................................................... 6Product characteristics of the Invisorb ® Spin Cell Mini Kit..................................................... 7Principle and procedure ............................................................................................................ 7Sampling and storage of starting material............................................................................... 7Procedure............................................................................................................................... 8Yield and quality of genomic DNA .......................................................................................... 8Important notes....................................................................................................................... 9Important points before starting a protocol.............................................................................. 9Preparing reagents and buffers .............................................................................................. 9Reagents and equipment to be supplied by user .................................................................. 10Important indications 10Scheme of the Invisorb ® Spin Cell Mini Kit: .......................................................................... 11Protocol 1: DNA isolation from cell culture (24-Well-Plates; max. 2x10 6 cells ....................... 12Protocol 2: DNA isolation from cell culture (96-Well-Plates)................................................. 13Protocol 3: DNA isolation of DNA from swab material (buccal swab; vaginal swab).............. 14Protocol 4: DNA isolation from Serum or Plasma (max. 100Al)............................................ 14Troubleshooting....................................................................................................................... 15Appendix................................................................................................................................... 16General notes on handling DNA ........................................................................................... 16Ordering information................................................................................................................ 17Invitek distributors ................................................................................................................... 182Invisorb ® Spin Cell Mini Kit 032010

Kit contents of the Invisorb ® Cell Mini KitStore all other kit components at room temperature (RT)!3 DNAextractions10 DNAextractions50 DNAextractions250 DNAextractionsCatalogue number 1033100100 1033100900 1033100200 1033100300Lysis Buffer D 5 ml 10 ml 30 ml125 mlBinding Buffer HL 2 ml 2 x 2 ml 15 ml 60 mlCarrier Suspension B 100 Al 150 Al 600 Al 2 x 1.3 mlWash Buffer15 ml(ready to use)15 ml(ready to use)18 ml(final volume 60 ml)2 x 45 ml(final volume 2 x 150 ml)Elution Buffer D 2 ml 2 x 2 ml 15 ml 60 mlRTA Spin Filter Set 3 10 50 2501.5 ml Receiver Tubes 3 10 50 250Manual 1 1 1 1Initial stepsWarmingwaterbath orthermomixer to70°C and heat theElution Buffer Dto the sametemperature.Warmingwaterbath orthermomixer to70°C and heat theElution Buffer Dto the sametemperature.Add 42 ml of 96-100% Ethanol to thebottle Wash Bufferand mix thoroughly.Store the WashBuffer always withfirmly closed cap.Storage at roomtemperature!Add 105 ml of 96-100%Ethanol to each bottleWash Buffer and mixthoroughly.Store the Wash Bufferalways with firmly closedcap.Storage at roomtemperature!Warming waterbathor thermomixer to70°C and heat theElution Buffer D tothe sametemperature.Warming waterbath orthermomixer to 70°Cand heat the ElutionBuffer D to the sametemperature.3Invisorb ® Spin Cell Mini Kit 032010

Symbolslot numbercatalog numberdate of manufactureexpiry dateconsult operating instructionstemperature limitationdo not reusemanufacturerStorage conditionsAll buffers and kit components of the Invisorb ® Spin Cell Mini Kit should be stored at roomtemperature (RT) and are stable for 12 months under these conditions.Room temperature (RT) is defined as range from 15 - 30°C.Wash Buffer charged with ethanol should be appropriately sealed and stored at roomtemperature.If there are any precipitates within the provided solutions dissolve these precipitates by carefullywarming up to room temperature (up to 30°C).Quality controlInvitek guarantees the correct function of the Invisorb ® Spin Cell Mini Kit for applications asdescribed in the manual. In accordance with Invitek’s certified Quality Management System allcomponents of the Invisorb ® Spin Cell Mini Kit were tested against predetermined specificationsto ensure consistent product quality.If you have any questions or problems regarding any aspects of Invisorb ® Spin Cell Mini Kit orother Invitek products, please do not hesitate to contact us.For technical support or further information please contact:from Germany +49-(0)30-9489-2901/ 2910from abroad +49-(0)30-9489-2907or contact your local distributor (see page 18).4Invisorb ® Spin Cell Mini Kit 032010

Intended useThe Invisorb ® Spin Cell Mini Kit, is the ideal tool for a fast and convenient manual isolationand purification of genomic DNA from cultured cells (24 or 96 well plates), serum, human andanimal lavage, fresh or frozen plasma, bone marrow and urine. For reproducible and highyields an appropriate sample storage is essential. The purified DNA can be used for in-vitrodiagnostic analysis.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 intended for use by professional users such as technicians, physicians, andbiologists trained in molecular biological techniques. It is designed to be used with anydownstream application employing enzymatic amplification or other enzymatic modification ofDNA followed by signal detection or amplification. Any diagnostic results generated using thesample preparation procedure in conjunction with any downstream diagnostic assay should beinterpreted with regard to other clinical or laboratory findings.To minimize irregularities in diagnostic results, adequate controls for downstream applicationsshould be used.Product use limitationThe kit is not validated for the isolation of genomic DNA bacteria, fungi, parasites, orpurification of RNA.The application of the kit for isolation and purification of viral DNA has not been evaluated.When changing the starting material or the flow trace, no guarantee in operability is issued.The user is responsible to validate the performance of the Invitek kits for any particular use, sincethe performance characteristics of our kits have not been validated for any specific application.Invitek kits may be used in clinical diagnostic laboratory systems after the laboratory has validatedthe complete diagnostic system as required by CLIA’ 88 regulations in the U.S. or equivalents inother countries.All products sold by Invitek are subjected to extensive quality control procedures according to ENISO 9001 : 2000 and EN ISO 13485 : 2003, and are warranted to perform as described whenused correctly. Any problems should be reported immediately.The chemicals and plastic parts are for laboratory use only, they have to be stored in thelaboratory and must not used for other purposes than intended.The kit contents are unfit for consumption.5Invisorb ® Spin Cell Mini Kit 032010

Safety informationWhen working with chemicals, always wear a suitable lab coat, disposable gloves, and protectivegoggles. Heed the legal requirements for working with biological material!For more information, please consult the appropriate material safety data sheets (MSDS). Theseare available in convenient and compact PDF format online at www.invitek.de under each Invitekkit and kit component.If the buffer bottles are damaged or leaking, wear gloves and protective goggles when discardingthe bottles in order to avoid any injuries.Invitek has not tested the liquid waste generated by the Invisorb ® Spin Cell Mini procedures forresidual infectious materials. Contamination of the liquid waste with residual infectious materials ishighly unlikely, but cannot be excluded completely. Therefore, liquid waste must be consideredinfectious and be handled and discarded according to local safety regulations.Below European Community risk and safety phrases for the components of the Invisorb ® Spin CellMini Kit to which they apply, are listed.Lysis Buffer D:Binding Buffer HL:Xn contains: Guanidinthiocyanate Xi , Fharmfulhighly flammable, irritant(R32, S2, S50)R11, 36, 67, S2, 7,16, S24/25/26R:11:highly flammableR36: irritating to eyesR67: vapours may cause drowsiness and dizzinessS2: keep out of the reach of childrenS7: keep container tightly closedS16: keep away from sources of ignition - No smokingS22: do not breath dustS24: avoid contact with skinS25: avoid contact with eyesS26: in case of contact with eyes, rinse immediately with plenty of water and seek medical adviceS50do not mix with strong acids or lyses, nonferrous heavy metals and their salts.Emergency medical information in english and german language can be obtained 24 hours a dayfrom:Poison Information Center Freiburg, Germany: Tel.: ++49 761-192406Invisorb ® Spin Cell Mini Kit 032010

Product characteristic of the Invisorb ® Spin Cell KitStarting materialYieldTime forpreparationRatio- max. 5 x 10 7 cells(2 ml of overnight culture )- cell cultures(max. 2 x 10 6 cells),- swabs, serum or plasmaup to 10 Ag of genomicDNA depends onamount and type ofstarting materialabout 20 - 30 min A 260 : A 2801.7 – 2.0The Invisorb ® Spin Cell Mini Kit has been designed for fast and easy purification of highmolecular-weightgenomic and mitochondrial DNA from cell culture, swabs, serum andplasma.After cell wall lysis the starting material will be directly mixed with an optimized Lysis BufferD including reagents inactivating intracellular DNases and also DNases found elsewhere inthe environment. DNA is on the surface of a filter as contaminants pass through. Remainingcontaminants and enzyme inhibitors are removed in an efficient wash steps and DNA is theneluted in water or buffer, ready for use.The Invisorb ® Spin Cell Mini Kit avoids the application of toxic cancerogeneous organicsolvents as well as ethanol precipitation. The convenient, purification procedure removescontaminants and enzyme inhibitors such as proteins and divalent cations, and purified DNAis ready for immediate use in sensitive downstream applications like, or for archiving.LLLLLPCR applicationsdigestion with restriction enzymesSouthern HybridizationRFLP analysisHLA typing (DQA; DQB).Principle and procedureThe Invisorb ® Spin Cell Mini Kit procedure comprises following steps:1. lysis of sample material2. binding the genomic DNA on a Spin Filter3. washing the Spin Filter and elimination of ethanol4. elution of genomic DNAThis manual contains 4 protocols, according to the different requirements of the startingmaterials.Sampling and storage of starting material:Cell cultures:The yield and quality of DNA may depend on factors such host strain, inoculation, antibiotic,and type of culture medium. The cells will be pelleted after cultivation. Best results areobtained 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, sincethis leads to reduced DNA size.7Invisorb ® Spin Cell Mini Kit 032010

Buccal swabs:To collect a sample, scrape the swab firmly against inside of each cheek 6 times. Air-dry theswab for at least 2h after collection or use them fresh prepared. Ensure that the personproviding the sample has not consumed any food or drink in the 30 min prior to samplecollection. Best results are obtained if the swab stays in the lysis solution during lysisprocedure. Use of poor quality starting material influences yield of purified DNA. This protocolis recommended for every common swab, like e.g. the following swab types: C:E:P: (OmniSwab from Whatman), cotton swab, Superswabs, Copan-Swab or DRACON tip fromHardwood Products company, CellProjects or Hain Diagnostika)Cells grown in suspension:Spin up to 1 x 10 7 cells for 5 min at 300 x g (1.500 rpm* ). Discard supernatant and remove allmedia completely, taking care not to disturb the cell pellet. At this point cells may be frozen (at -20°C or - 80°C) for future use or may be used immediately.Cells grown in a monolayer:Aspirate the media completely from the cells and continue immediately with the lysis step.Alternatively cells can be detached by trypsination (cultivation in larger culture vessels, likedishes > 35 mm, flasks > 12.5 cm 2 ). Transfer cells to a 50 ml reaction tube, pellet bycentrifugation at 300 x g (1.500 rpm* ) for 5 min and aspirate supernatant completely.ProcedureLysisSamples are lysed at elevated temperatures. Lysis is performed in the presence of LysisBuffer D.Binding genomic DNABy adding Binding Buffer HL to the lysate, optimal binding conditions will be adjusted.All lysate is applied to a spin filterRemoving residual contaminationsContaminants are efficiently washed away using Wash Buffer, while the genomic DNAremains bound to the spin filter.ElutionGenomic DNA is eluted from the spin filter using 30 - 200 Al Elution Buffer D. The eluted DNAis ready for use in different downstream applications. Eluted DNA stored at 4 – 8°C is stable for2 months or for more than 5 years if stored at -20°C.Yield and quality of genomic DNAThe amount of purified DNA using the Invisorb ® Spin Cell Mini Kit procedure, depends on thestrain, the number of cells in the sample, the sample source, transport conditions, storage, andage of the sample.Yield and quality of isolated genomic DNA is suitable for any molecular-diagnostic detectionsystem. The diagnostic tests should be performed according to manufacturers’ specifications.8Invisorb ® Spin Cell Mini Kit 032010

Important notesImportant points before starting a protocolAfter receiving the kit, check the kit components for damage. If buffer bottles are damaged,contact the Invitek Technical Services or your local distributor. In case of liquid spillage, refer to“Safety information” (page 6). Do not use damaged kit components, since their use may lead topoor kit performance.LLLLLLLLalways change pipet tips between liquid transfer. To avoid cross-contamination, werecommend the use of aerosol-barrier pipet tipsall centrifugation steps are carried out at room temperaturewhen working with chemicals, always wear a suitable lab coat, disposable gloves, andprotective gogglesdiscard gloves if they become contaminateddo not combine components of different kits unless the lot numbers are identicalavoid microbial contamination of the kit reagentsto minimize the risk of infections from potentially infectious material, we recommendworking under laminar air-flow until the samples are lysedthis kit should only be used by trained personalPreparing reagents and buffers1. adjust the thermo mixer to 37°C and 70°C.2. warm up the needed amount of Elution Buffer D to 70°C(100 - 200 Al Elution Buffer D are needed per sample).3. label the needed amount of Spin Filter4. label the needed amount of 1.5 ml Receiver Tubes5. add the needed amount of ethanol to the Wash Buffer3 DNA or 10 extractions:Wash Buffer is ready to use!50 DNA extractions:Add 42 ml of 96 - 100 % ethanol to Wash Buffer, mix thoroughly and always keep the bottlefirmly closed!250 DNA extractions:Add 105 ml of 96 - 100 % ethanol to Wash Buffer, mix thoroughly and always keep the bottlefirmly closed!9Invisorb ® Spin Cell Mini Kit 032010

Reagents and equipment to be supplied by userWhen working with chemicals, always wear a suitable lab coat, disposable gloves, and protectivegoggles. For more information, please consult the appropriate material safety data sheets(MSDS). These are available online in convenient and compact PDF format at www.invitek.deunder each Invitek kit and kit component.LLLLLLLLmicro centrifugethermo mixer (for 70°C)measuring cylinder (250 ml)disposable glovespipette and pipette tipsvortexerreaction tubes (1.5 ml or 2.0 ml)dd H 2 O96 - 100 % ethanolImportant indications1. Process only as much samples as the micro centrifuge allows to process.2. Buffers should be thoroughly mixed and should have room temperature3. The elution can be done by using lower amount of Elution Buffer D. This may result in ahigher concentration of DNA. But pay attention that minimum volume for elution is 50 Al,but this will reduce the yield. Elution volume between 2 x 50 Al up to 200 Al will realizecomparable results.4. The eluted DNA volume can be lower than the added Elution Buffer D volume.Elution Buffer D should be preheated to 56 °C.5. The Elution Buffer D doesn’t contain EDTA.6. The yield can be increased, if the incubation time with preheated Elution Buffer D will beprolonged.10Invisorb ® Spin Cell Mini Kit 032010

Scheme of the Invisorb ® Spin Cell KitPlease read protocols prior the start of the preparation carefully--------------------------------------------------------------------------------------remove medium and wash cells twice with 1 x PBS (not included in the kit)add 500 Al Lysis Buffer D pipet up and down several times and incubateat room temperature for 3 min.transfer the whole cell lysis suspension into a 1.5 ml or 2.0 ml reaction tube.add 10 Al Carrier Suspension B and 200 Al Binding Buffer HLmix briefly and transfer the suspension directly onto the RTA Spin Filter.incubate for 2 mincentrifuge for 3 min at 13.000 x g (12.000 rpm)discard the flow-through and reuse the RTA Receiver Tube.Add 550 Al Wash Buffer and centrifuge for 1 min at 13.400 x g (12.000 rpm).Discard the flow-throughrepeat step once again.centrifuge for 3 min at 13.400 x g (12.000 rpm) toremove the residual ethanol.transfer the RTA Spin Filter into a new 1.5 ml Receiver Tube.genomic DNAadd 200 Al of Elution Buffer D prewar med to 70°C directly onto themembrane of the RTA Spin Filter.incubate for 2 min.centrifuge at 9.300 x g (10.000 rpm) for 2 min.discard the Spin Filterclose the 1.5 ml Receiver Tube and store the eluted DNA sample at 4 °C11Invisorb ® Spin Cell Mini Kit 032010

Protocol 1: DNA isolation from cell culture (24-Well-Plates;max. 2x10 6 cells)Important: Incubate the Elution Buffer D at 70°C1. Remove medium and wash cells twice with 1 x PBS (not included in the kit).2. Add 500 Al Lysis Buffer D per well, pipet up and down several times and incubate at roomtemperature for 3 min.Note:Should Lysis Buffer D contain any precipitates incubate it in a 50°C waterbath for a short time!3. Transfer the whole cell lysis suspension into a 1.5 ml or 2.0 ml microcentrifuge tube.Note:Vortex the Carrier Suspension B thoroughly prior to use.4. Addition of 10 Al Carrier Suspension B and 200 Al Binding Buffer HL, mix briefly andtransfer the suspension directly onto the RTA Spin Filter. Incubation for 2 min.5. Centrifugation for 3 min at 13.400 x g (12.000 rpm). Discard the flow-through and reuse theRTA Receiver Tube.6. Add 550 Al Wash Buffer and centrifuge for 1 min at 13.400 x g (12.000 rpm). Discard theflow-through.7. Repeat step 6 once again. Afterwards centrifuge for 3 min at 13.400 x g (12.000 rpm) toremove the residual ethanol.8. Transfer the RTA Spin Filter into a new 1.5 ml Receiver Tube. Add 200 Al of ElutionBuffer D or Tris buffered ddH 2 0 (pH 8.5 - 9.0) prewarmed to 70°C directly onto themembrane of the RTA Spin Filter.Incubation for 2 min. Centrifugation at 9.300 x g (10.000 rpm) for 2 min.Discard the RTA Spin Filter.12Invisorb ® Spin Cell Mini Kit 032010

Protocol 2: DNA isolation from cell culture (96-Well-Plates)Important: Incubate the Elution Buffer D at 70°C1. Remove medium and wash cells twice with 1 x PBS (not included in the kit).2. Add 350 Al Lysis Buffer D per well, pipet up and down several times and incubate at roomtemperature for 5 min.Note:Should Lysis Buffer D contain any precipitates incubate it in a 50°C waterbath for a short time!3. Transfer the whole cell lysis suspension into a 1.5 ml or 2.0 ml microcentrifuge tube.Note:Vortex the Carrier Suspension B thoroughly prior to use.4. Addition of 10 Al Carrier Suspension B, 150 Al Lysis Buffer D and 200 Al Binding BufferHL, mix briefly and transfer the suspension directly onto the RTA Spin Filter.Incubation for 2 min.5. Centrifugation for 3 min at 13.400 x g (12.000 rpm). Discard the flow-through and reuse theRTA Receiver Tube.6. Add 550 Al Wash Buffer and centrifuge for 1 min at 13.400 x g (12.000 rpm). Discard theflow-through.7. Repeat step 6 once again. Afterwards centrifuge for 3 min at 13.400 x g (12.000 rpm) toremove the residual ethanol.8. Transfer the RTA Spin Filter into a new 1.5 ml Receiver Tube. Add 100 Al of ElutionBuffer D or Tris buffered ddH 2 0 (pH 8.5 - 9.0) prewarmed to 70°C directly onto themembrane of the RTA Spin Filter. Incubation for 2 min. Centrifugation at 9.300 x g(10.000 rpm) for 2 min. Discard the RTA Spin Filter.13Invisorb ® Spin Cell Mini Kit 032010

Protocol 3: DNA isolation of DNA from Swab material(Buccal Swab; Vaginal Swab)Important: Incubate the Elution Buffer D at 70°C1. Incubation of the swab in a 1.5 or 2.0 ml microfuge tube with 500 Al Lysis Buffer D for 5 min.Grind the swab on the wall of the tube carefully and remove the swab.Note: Should Lysis Buffer D contain any precipitates incubate it in a 50°C waterbath for a short time!Note:Vortex the Carrier Suspension B thoroughly prior to use.2. Addition of 10 Al Carrier Suspension B and 200 Al of Binding Buffer HL, mix briefly andtransfer the suspension directly onto the RTA Spin Filter. Incubation for 2 min.3. Centrifugation for 3 min at 13.400 x g (12.000 rpm). Discard the flow-through and reuse theRTA Receiver Tube.4. Addition of 550 Al Wash Buffer and centrifugation for 1 min at 13.400 x g (12.000 rpm).Discard the flow-through.5. Repeat step 4 once again. Afterwards centrifuge for 3 min at 13.400 x g (12.000 rpm) toremove the residual ethanol.6. Transfer the RTA Spin Filter into a new 1.5 ml Receiver Tube. Add 100 Al of ElutionBuffer D or Tris buffered ddH 2 0 (pH 8.5 - 9.0) prewarmed to 70°C directly onto themembrane of the RTA Spin Filter. Incubation for 2 min. Centrifugation at 9.300 x g(10.000 rpm) for 2 min.Discard the RTA Spin Filter.Protocol 4: DNA isolation from Serum or Plasma (max. 100Fl)Important: Incubate the Elution Buffer D at 70°C1. Addition of 500 Al Lysis Buffer D to the Serum or Plasma sample (max. 100 Al) andincubation for 10 min (continuous shaking increases the lysis efficiency!).Note:Note:Should Lysis Buffer D contain any precipitates incubate it in a 50°C waterbath for a shorttime!Vortex the Carrier Suspension B thoroughly prior to use.2. Addition of 10 Al Carrier Suspension B and 200 Al Binding Buffer HL, mix briefly andtransfer the suspension directly onto the RTA Spin Filter. Incubation for 2 min.3. Centrifugation for 3 min at 13.400 x g (12.000 rpm). Discard the flow-through and reuse theRTA Receiver Tube.4. Addition of 550 Al Wash Buffer and centrifugation for 1 min at 13.400 x g (12.000 rpm).Discard the flow-through.5. Repeat step 4 once again. Afterwards centrifuge for 3 min at 13.400 x g (12.000 rpm) toremove the residual ethanol.6. Transfer the RTA Spin Filter into a new 1.5 ml Receiver Tube. Add 100 Al of ElutionBuffer D or Tris buffered ddH 2 0 (pH 8.5 - 9.0) prewarmed to 70°C directly onto themembrane of the RTA Spin Filter. IIncubation for 2 min. Centrifugation at 9.300 x g(10.000 rpm) for 2 min. Discard the RTA Spin Filter.14Invisorb ® Spin Cell Mini Kit 032010

TroubleshootingProblem Cause Comments and suggestionslow yield ofDNAincomplete lysis of startingmaterialimproper storage of startingmaterialpoor DNA binding of the DNAsubstantial loss of boundDNA in the washing step dueto low ethanol concentrationin the Wash Bufferpoor elution of the DNAincrease the lysis timestore the samples carefully.mix the sample with the Binding Buffer HLcarefully.prepare the Wash Buffer exactly as describedin the manualstorge of Wash Buffer with firmly fixed capcarry out a second elutionprewarm the Elution medium to 70°Cclotting of themembranestarting volume to highcentrifugation speed to slowreduce starting volumeincrease the Centrifugation speedincrease the Centrifugation timebad results inPCRcontamination of final DNAsolution with residual saltscontamination of final DNAsolution by ethanolwashing of the Spin Filter as described inmanualremove the ethanol careful (test the smell)poor cleavage byrestrictionendonucleasesEDTA in the Elution Buffer Delution of DNA in Tris buffered dd H 2 O(especially when using enzymes sensitive toEDTA).15Invisorb ® Spin Cell Mini Kit 032010

AppendixGeneral notes on handling DNANature of DNAThe length and delicate physical nature of DNA requires careful handling to avoid damagedue to shearing and enzymatic degradation. Other conditions that affect the integrity andstability of DNA include acidic and alkaline environments, high temperature, and UVirradiation. Careful isolation and handling of high molecular weight DNA is necessary toensure compatibility with various downstream applications. Damaged DNA could performpoorly in applications such as genomic Southern blotting, and long-template PCR.Storage of DNAA working stock of DNA can be stored at 2 – 4RC for several weeks. For long term storageDNA should be stored at -20RC, but storing at – 20°C can cause shearing, particularly if theDNA is exposed to repeated freeze-thaw cycles.Note that the solution in which the nucleic acid is eluted in will affect it’s stability duringstorage. 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 DNAAvoid over drying genomic DNA after ethanol precipitation. It is better to let it air dry than touse a vacuum, although vacuum drying can be used with caution.Avoid vigorous pipetting. Pipetting genomic DNA through small tip openings causes shearingor nicking. One way to decrease shearing of genomic DNA is to use special tips that havewide openings designed for pipetting genomic DNA.DNA YieldThe amount of purified DNA from the cells, depends on the DNA content of the sample,transport, storage, and age.16Invisorb ® Spin Cell Mini Kit 032010

Ordering informationProduct Package size Order-Nr.Invisorb ® Spin Cell Mini Kit 3 preparations 1033100100Invisorb ® Spin Cell Mini Kit 50 preparations 1033100200Invisorb ® Spin Cell Mini Kit 250 preparations 1033100300Single components for Invisorb ® Cell Mini KitLysis Buffer D 30 ml 1033101000Binding Buffer HL 10 ml 1033102300Carrier Suspension B 0.5 ml 1033107200Wash Buffer (add 42 ml) 18 ml 1033103200Elution Buffer D 15 ml 1033104000Related productsInvisorb ® Spin Yeast DNA Mini Kit 3 preparations 1033120100Invisorb ® Spin Yeast DNA Mini Kit 50 preparations 1033120300Invisorb ® Spin Yeast DNA Mini Kit 250 preparations 1033120400Invisorb ® Spin Swab Kit 3 preparations 1035120100Invisorb ® Spin Swab Kit 50 preparations 1035120200Invisorb ® Spin Swab Kit 250 preparations 1035120300PSP ® SalivaGene DNA Kit 3 preparations 1035200100PSP ® SalivaGene DNA Kit 50 preparations 1035200200PSP ® SalivaGene DNA Kit 250 preparations 1035200300Invisorb ® Spin Tissue Mini Kit 3 preparations 1032100100Invisorb ® Spin Tissue Mini Kit 50 preparations 1032100200Invisorb ® Spin Tissue Mini Kit 250 preparations 1032100300Invisorb ® Tissue HTS 96 Kit/C 2 x 96 preparations 7032300200Invisorb ® Tissue HTS 96 Kit/C 4 x 96 preparations 7032300300Invisorb ® Tissue HTS 96 Kit/C 24 x 96 preparations 7032300400Invisorb ® DNA Cell HTS 96 Kit/C 2 x 96 preparations 7039400200Invisorb ® DNA Cell HTS 96 Kit/C 4 x 96 preparations 7039400300Invisorb ® DNA Cell HTS 96 Kit/C 24 x 96 preparations 7039400400InviMag ® Tissue DNA Kit/ KF96 1 x 96 preparations 7432300100InviMag ® Tissue DNA Kit/ KF96 5 x 96 preparations 7432300200InviMag ® Tissue DNA Kit/ KFmL 1 x 75 preparations 2432110100InviMag ® Tissue DNA Kit/ KFmL 5 x 75 preparations 243211010017Invisorb ® Spin Cell Mini Kit 032010

Invitek distributorsAustraliaMedica Pacifica Ltdbhoo@medica.co.nzwww.medica.co.nzFreephone: 1800 201 386Mobile: 021 633 820Phone: ++ 649 629 0823Fax: ++ 649 629 0542AustriaIBL Martinibl@ibl.or.atwww.ibl.or.atPhone:++ 43 02246-20 500Fax.:++ 43 02246-20 500 20BrazilUNISCIENCE DO BRASIL Ind. Com. Rep. Ltdasergio.joelsons@uniscience.comwww.uniscience.com.brPhone: ++ 55 (11) 3622-2328Fax ++ 55 (11) 3622-2333BelgiumWestburg b.v.info@westburg.nlwww.westburg.nlPhone:++ 31 33 4950094Fax.:++ 31 33 4951222CanadaESBE Scientificinfo@esbe.comwww.esbe.comPhone:++ 1 800-268-3477Fax: ++ 1-800-387-0476ChinaGene Tech (Shanghai) Co. Ltd6/F, Building 25, No 69 Guiqing Road,Cao He-jing High-Tech Park,Shanghai 200233P.R.ChinaTel: ++ 86-021-64957570Fax: ++ 86-021-64957610ChileAndes Importadora y Exportadora Ltdacristian.candia@andesimport.clwww.andesimport.clPhone:++ 56 - 2 – 6834510Fax: ++ 56 - 2 - 6834510Czech RepublicCHEMOS Cz, s.r.o.info@chemos.czwww.chemos.czPhone:++ 420 272 701 793Fax:++ 420 272 705 485Czech RepublicMERCI, s.r.o.Hviezdoslavova 55bjanecka@merci.czwww.merci.czPhone:++ 420 602 410 197DenmarkWestburg b.v.info@westburg.nlwww.westburg.nlPhone: ++ 45 48 76 06 93/ ++ 31 33 4950094Fax: ++ 45 30 62 33 34/ ++ 31 33 4951222EstoniaICOSAGEN ASNooruse 950411 Tartuinfo@icosagen.comwww.icosagen.comPhone:++ 372 737 7070FinlandWestburg b.v.info@westburg.nlwww.westburg.nlPhone: ++ 45 48 76 06 93/ ++ 31 33 4950094Phone: ++ 45 30 62 33 34 / + 31 33 4951222FranceLaboratoire EUROBIOadv@eurobio.frwww.eurobio.frPhone: ++ 33(0)1 60 92 21 83Fax.:++ 33(0) 1 69 07 95 34GreeceBioline Scientificdemagos@hol.grPhone:++ 3015226547 / ++ 3015249881Fax.:++ 30 1 5244744Hong KongB & R Technology Ltd.leo_law@bnr.com.hkPhone:++ 852 2547 8688Fax.:++ 852 2915 1522IcelandWestburg b.v.info@westburg.nlwww.westburg.nlPhone:++ 31 33 4950094Fax.:++ 31 33 4951222IndiaGenxBio Health Sciences Pvt. Ltd,.genxbio@gmail.comwww.genxbio.comPhone:++919216502501Fax.:++911-55607726IranAria Exirm.b@ariaexir.comwww.ariaexir.comPhone:++ 98 21 8872 1001Fax.: ++ 98 21 8872 1001IsraelGadot Laboratory Supply LTDayelet@gadot.comPhone:++ 972 -50-7744842Fax.:++972 -8-6900650ItalySPA Società Prodotti Antibioticiceroni@spaspa.itwww.spaspa.it/biospa.htmPhone:++ 39 02 8913 9542Fax.:++ 39 02 8912 0996JapanWako Pure Chemicals Industries, Ltd.satoh.yumiko@wako-chem.co.jpwww.wako-chem.co.jpPhone:++ 81-6-6203-374Fax.:++ 81-6-6229-129EgyptGenoLab co. for importing & tradinggeno_lab@hotmail.comPhone:++ 20 012 7307778Fax.:++ 20 02 670647018 Invisorb ® Spin Cell Mini Kit 032010

KoreaPhilekorea Technology Inc.www.philekorea.co.krPhone: ++ 82-42-862-9636Fax: ++ 82-42-862-9638LuxembourgWestburg b.v.info@westburg.nlwww.westburg.nlPhone:++ 31 33 4950094Fax.:++ 31 33 4951222NetherlandsWestburg b.v.info@westburg.nlwww.westburg.nlPhone:++ 31 33 4950094Fax.:++ 31 33 4951222New ZealandMedica Pacifica Ltdbhoo@medica.co.nzwww.medica.co.nzFreephone: 1800 201 386Mobile: 021 633 820Phone: ++ 649 629 0823Fax: ++ 649 629 0542NorwayWestburg b.v.info@westburg.nlwww.westburg.nlPhone: ++ 45 30 62 33 34 /++ 31 33 4950094Fax.:++ 31 33 4951222PolandBiotcom Polskaoffice@biotcom.plPhone:++ 48 71 363 24 82Fax.:++ 48 71 363 12 63PortugalQUILABAN - Química Laboratorial Analítica, Lda.quilaban@quilaban.ptwww.quilaban.ptPhone:++ 351 21 923 63 50Fax.:++ 351 21 923 63 89RomaniaVitroBioChem SRLvbc_office@yahoo.comPhone: ++ 40 21 411 60 23Fax.: ++ 40 21 411 60 23RussiaHelicon Company Ltd.kirillov@helicon.ruwww.helicon.ruPhone: ++7-495-933-27-36Fax: ++7-495-930-00-84SloveniaSanolabor, d.d.katja.sifrer@sanolabor.siwww.sanolabor.siPhone: ++ 386 (0)1 585-4211Fax: ++ 386 (0)1 524-9033South Africa & SimbabweCeltic Molecular Diagnosticdermot@celticdiagnostics.comwww. celticdiagnostics.comPhone: ++ 27 21 685 1112Fax: ++27 21 685 1117SpainNucliberinfo@nucliber.comwww.nucliber.comPhone:++ 34 91 5062940Fax.:++ 34 91 5394330SyriaNEW MED TECHNOLOGYDamascus - Syrianew_medtech@yahoo.comPhone: ++ 96311-88271717Fax:++96311- 88271710SwedenWestburg b.v.info@westburg.nlwww.westburg.nlPhone: ++ 45 48 76 06 93 / ++ 45 30 62 33 34Fax.:++ 31 33 4951222SwitzerlandChemie Brunschwig AGinfo@brunschwig-ch.comwww.brunschwig-ch.comPhone: ++ 41 (0)61 308 91 11Fax: ++ 41 61 308 91 19TaiwanPrisma Biotech Co.product@prismabiotech.com.twwww. prismabiotech.com.twPhone:++ 886 2 2723 0886Fax.:++ 886 2 2723 0876ThailandPrima Scientific Co.,Ltdprimasci@primasci.comwww.primasci.comTel: ++ 66(0) 2435-7434,/2434-3771/2433-2081Fax: ++ 66(0) 2884-6441/2597-5209United KingdomThistle Scientificinfo@thistlescientific.co.ukwww.thistlescientific.co.ukPhone: ++ 44 1698 338844Fax.:++ 44 1698 338880United StatesB-Bridge International, Inc.paul@b-bridge.comhttp://www.b-bridge.comPhone:++ (1) 650-969-7727Fax:++ (1) 650-969-7737VenezuelaBiochrom camiguelbio@cantv.netwww.biochrom.com.vePhone:++ 58 212 975 2617, 976 3079Fax:++58 212 978 4180VietnamPrima Scientific Co.,Ltdprimasci@primasci.comwww.primasci.comTel: ++66 (0) 2435-7434,/2434-3771/2433-2081Fax: ++66 (0) 2884-6441/2597-5209VietnamVIET HUY Technological Scientific Equipment Co., Ltd48 Cu Lao St, Ward 02Phu Nhuan District, HCMC, VietnamTel: ++ 84 (8) 5449 2277/ 78Fax: ++ 84 (8) 5449 227918 Invisorb ® Spin Cell Mini Kit 032010