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Invisorb Fragment CleanUp

1. components contamination with agarose traces contamination of the final DNA with ethanol prepare the Wash Buffer exactly as described in the manual storage of Wash Buffer with firmly fixed cap add the Elution Buffer directly onto the centre of the Spin Filter even if a small elution volume is used the binding of DNA fragments under TBE buffer condition is slightly reduced for smaller fragments than 500 bp please use TAE agarose gels the gel slice must be completely dissolved add the amount of Binding Enhancer needed to the solubilized suspension Washing of the Spin Filters as described in the manual prolong the incubation time with Wash Buffer to 5 minutes before centrifugation wash the Spin Filter one time with Gel Solubilizer S keep the given centrifugation time extend it if necessary test the smell 24 PCR product purification DNA Fragment Cleanup 0515 Ordering information Product Package Size Catalogue No MSB Spin PCRapace 50 purifications 1020220200 MSB Spin PCRapace 250 purifications 1020220300 MSB Spin PCRapace 500 purifications 1020220400 Single components Elution Buffer 15 ml 1020224000 Invisorb Spin DNA Extraction Kit 50 purifications 1020110200 Invisorb Spin DNA Extraction Kit 250 purifications 1020110300 Single components Gel Solubilizer 30 ml 1020118000 Wash Buffer add 42ml ethanol 18 ml 1020113000 Elution Buffer 15 ml 1020114000 Invisorb Fragment CleanU
2. 0515 Special Protocol 3 Purification and concentration of PCR products from 200 ul PCR reactions Please read the instructions carefully and conduct the prepared procedure for MSB Spin PCRapace and Invisorb Fragment CleanUp Note Before starting with the purification procedure please place a Spin Filter into a 2 0 ml Receiver Tube Attention Please be aware that you have to prepare the Binding Buffer see instruction page 14 1 Binding of the PCR fragments For PCR mixture 200 ul Add 1000 ul Binding Buffer to the PCR sample and mix very well by pipetting or vortexing Transfer the sample in two aliquots onto a Spin Filter and centrifuge for 2 min at 11 000 x g 11 000 rpm Remove the filtrate and centrifuge again for 3 minutes 2 Elution of the PCR fragments Place the Spin Filter into a new 1 5 ml Receiver Tube Add at least 10 ul Elution Buffer or ddH 0 directly onto the center of the Spin Filter Incubate for 1 minute at room temperature Centrifugation for 1 minute at 11 000 x g 11 000 rpm Important Notes 1 The provided volume of Binding Buffer is calculated based on the needed buffer volumes in protocol 1 and 2 The needed amount of protocol 3 is not considered You can order additional Binding Buffer Cat No 10202220 for this application by phone 49 0 30 948928910 01 or by mail 2 If the PCR mixture contains mineral oil we recommend the addition of 500 pl of Binding Buffer independent of
3. High concentrated pure DNA fragments are eluted with a minimum of 10 ul of Tris buffer or water Maximum elution efficiency is achieved between pH 7 0 and 8 5 therefore when using water to elute be certain that the pH is within this range DNA must also be stored at 20 C when eluted with water as it is possible that DNA may degrade without a buffering agent DNA yield and concentration DNA yield depends on the following three factors the volume of elution buffer how the buffer is applied to the column and the incubation time of the buffer on the column Between 100 and 200 ul of the elution buffer completely covers the Invisorb membrane ensuring maximum yield even when not applied directly to the center of the membrane Elution with up to 50 ul requires the buffer to be added directly to the center of the membrane and an additional 1 2 minute incubation is required for maximum yield if elution is performed with the minimum recommended volume of 10ul Preparing reagents and buffers of the Invisorb Spin DNA Extraction Kit 10 preps add 8 ml 99 7 Isopropanol to the Binding Enhancer 50 preps add 42 ml 96 100 ethanol to the bottle Wash Buffer add 24 ml 99 7 Isopropanol to the Binding Enhancer 250 preps add 105 ml 96 100 ethanol to each bottle Wash Buffer add 40 ml 99 7 Isopropanol to each Binding Enhancer 16 PCR product purification DNA Fragment Cleanup 0515 General Scheme of Invisorb Sp
4. OW co 20 21 22 23 23 24 25 PCR product purification DNA Fragment Cleanup 0515 Kit contents of the MSB Spin PCRapace Store all kit components at room temperature Binding Buffer final volume 11 ml 10 preps 50 preps 250 preps 500 preps Catalogue No 1020220900 1020220200 1020220300 1020220400 4 ml 12 ml 63 ml 2x 63 ml final volume 32 ml final volume 163 ml final volume 2 x 163 ml Isopropanol to the Binding Buffer Isopropanol to the Binding Buffer Elution Buffer 2ml 3x2ml 30 ml 60 ml Spin Filter 10 50 5x50 10x50 2 0 ml Receiver Tubes 10 50 5 x 50 10 x 50 1 5 ml Receiver Tubes 10 50 5 x 50 10 x 50 Manual 1 1 1 1 Initial steps add 7 ml 99 7 add 20 ml 99 7 add 100 ml 99 7 add 100 ml 99 7 Isopropanol to the Opt Isopropanol to each Binding Buffer Binding Buffer Kit contents of the Invisorb Spin DNA Extraction Kit Store all kit components at room temperature 5 preps 50 preps 250 preps Catalogue No 1020110100 1020110200 1020110300 Gel Solubilizer S 3x2ml 60 ml 2x 140 ml 2ml 6 ml 3x 10 ml Binding Enhancer final volume 10 ml final volume 30 ml final volume 50 ml Wash Buffer 15 ml ready to use 18 ml final volume 60 ml 2x 45 ml final volume 2 x 150 ml Elution Buffer 2ml 2x2ml 15 ml Spin Filter 5 50 5x50 2 0 ml Receiver Tubes 5 50 5x50 1 5 ml Receiver Tubes 5 50 5x5
5. a convenient tool for ultra fast and efficient direct purification of about 100 ul PCR products from 80 bp up to 30 kb from amplification reactions With max 5 min of processing time the MSB Spin PCRapace is the fastest system for the separation of PCR products 80 bp 30 kb from dNTP s primers enzymes additives labelling reagents biotin radioactive ATP etc and salts The recovery of PCR product is 80 95 The kit is further useful for DNA fragment cleanup from o restriction digestion mixture o ligation mixture o CDNA synthesis mixtures The kit is also a powerful and efficient tool for the o concentration of DNA fragments as well as o for purification of linearized pDNA from restriction mixtures and o for the reliable removal of Dye terminators from DNA cycle sequencing reactions The DNA fragments will be bound directly onto the surface of a spin filter column based on new buffer composition No additional and common used washing steps are necessary Finally the DNA fragments will be eluted with low salt buffer or H20 Binding capacity for fragment size of 200 500 bp up to 10 ug Binding capacity for fragment size of 20 000 kb up to 100 ug Elution volume minimum 10 ul Beside the extremely time efficient procedure the kits provides high yield and purity of the recovered DNA fragments The recovery rate 80 95 is nearly independent from PCR additives the PCR reaction volume and the fragment size The purified PC
6. and ethanol o elution of highly pure DNA fragment or PCR product Binding of DNA fragments The reaction mixture is mixed in a ratio of 1 5 with the Binding Buffer to provide the appropriate condition for the binding of DNA fragments in range of 70 bp 30 kb to the silica membrane under minimal concentrations of non chaotropic salts The binding of small DNA fragments can be supported by the addition of small amounts of isopropanol but this ratio is very sensitive Removal of Contaminants The DNA fragments bind to the membrane at minimal concentrations of non chaotropic salts Therefore a washing step is not required Unwanted primers and impurities such as salts enzymes unincorporated nucleotides dyes ethidium bromide oils and detergents do not bind to the silica membrane instead are drawn through the column by centrifugal force together with the big access of Binding Buffer Any remaining Binding Buffer which may interfere with subsequent enzymatic reactions is removed by additional centrifugation Elution of PCR products or DNA fragments The efficiency of elution strongly depends on the concentration of salt and on the pH of the elution buffer Unlike absorption elution is most efficient under basic conditions and low concentrations of salt High concentrated pure DNA fragments are eluted with a minimum of 10 ul of Tris buffer or water Maximum elution efficiency is achieved between pH 7 0 and 8 5 therefore when using water
7. discarding the bottles in order to avoid any injuries STRATEC Molecular has not tested the liquid waste generated by the MSB Spin PCRapace Invisorb Spin DNA Extraction Kit and the Invisorb Fragment CleanUp 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 must be handled and discarded according to local safety regulations European Community risk and safety phrases for the components of the MSB Spin PCRapace Invisorb Spin DNA Extraction Kit and the Invisorb Fragment CleanUp to which they apply are listed below as follows Gel Solubilizer warning H302 312 332 412 EUH032 P273 H302 Harmful if swallowed H312 Harmful in contact with skin H332 Harmful if inhaled H412 Harmful to aquatic life with long lasting effects EUH032 Contact with acids liberates very toxic gas present and easy to do Continue rinsing P273 Avoid release to the environment Emergency medical information can be obtained 24 hours a day from infotrac outside of USA 1 352 323 3500 in USA 1 800 535 5053 Intended use See for each product the product characteristic there are presented the intended use and the product use limitation for each kit THE PRODUCT IS INTENDED FOR USE BY PROFESSIONALS ONLY SUCH AS TECHNICIANS PHYSICIANS AND BIOLOGIS
8. eluted in a low salt buffer after washing The extraction protocol as well as all buffers is optimized to provide high yield and purity of the recovered DNA fragment The hands on time necessary for the whole procedure is reduced to a minimum The purification process will be ready in 5 20 minutes The purified DNA fragments are ready to use in various downstream applications such as o Digestion with restriction enzymes o Invitro Transcription o Hybridization o Ligation and transformation o Labelling o Radioactive and fluorescent sequencing o Cloning o Amplification o Sequencing o Microinjection Preparing reagents and buffers of the Invisorb Fragment CleanUp 10 preps add 8 ml 99 7 Isopropanol to the Binding Enhancer add 7 ml 99 7 Isopropanol to the Binding Buffer 50 preps add 42 ml 96 100 ethanol to the bottle Wash Buffer add 24 ml 99 7 Isopropanol to the Binding Enhancer add 20 ml 99 7 Isopropanol to the Binding Buffer 250 preps add 105 ml 96 100 ethanol to each bottle Wash Buffer add 120 ml 99 7 Isopropanol to the Binding Enhancer add 100 ml 99 7 Isopropanol to the Binding Buffer 18 PCR product purification DNA Fragment Cleanup 0515 General Scheme of Invisorb Fragment CleanUp PCR cleanup Agarose gel extraction Solubilized gel slice Adjustment of binding conditions Adjustment of gt binding ar conditions Binding of PCR product AX Binding of the DNA f
9. 0 C for 10 minutes until the agarose gel slice is completely dissolved Incubation under continuous shaking e g Eppendorf Thermo mixer is very helpful 3 Add 250 ul Binding Enhancer to a 500 ul reaction volume or 500 ul Binding Enhancer to a 1 ml reaction volume and mix the suspension by pipetting some times or by vortexing Load approx 800 ul of the sample onto the Spin Filter Centrifuge at 11 000 x g 11 000 rpm for 2 minute Discard the filtrate For reaction volumes gt 800 ul reload the residual volume onto the Spin Filter and repeat the centrifugation step 4 Add 500 ul Wash Buffer to the Spin Filter and centrifuge for 1 min at 11 000 x g 11 000 rpm Discard the filtrate Repeat the washing step once again 5 Discard the filtrate Remove the residual ethanol of the Wash Buffer by centrifugation for 4 min at maximum speed 6 Transfer the Spin Filter into a new 1 5 ml Receiver Tube Add at least 20 ul Elution Buffer directly onto the center of the Spin Filter Incubate at room temperature for 5 minutes Centrifuge for 1 minute at 11 000 x g 11 000 rpm Note To increase the final DNA yield we recommend using a higher volume of Elution Buffer Please take into account that an increasing volume of Elution Buffer reduces the final concentration of the purified DNA An extended incubation time with Elution Buffer up to 10 minutes leads also to a slightly higher final yield Protocol 5 Instruction for repair of A overhangs in
10. 0 Manual 1 1 1 Initial steps add 8 ml 99 7 add 24 ml 99 7 add 40 ml 99 7 Isopropanol to the Binding Enhancer Isopropanol to the Binding Enhancer add 42 ml 96 100 ethanol to the bottle Wash Buffer Isopropanol to each Binding Enhancer add 105 ml 96 100 ethanol to each bottle Wash Buffer PCR product purification DNA Fragment Cleanup 0515 Kit contents of Invisorb Fragment CleanUp Store all kit components at room temperature 10 preps 50 preps 250 preps Catalogue No 1020300900 1020300200 1020300300 Gel Solubilizer S 12 ml 60 ml 2x 140 ml ape 4 ml 12 ml 63 ml Binding Buffer final volume 11 ml final volume 32 ml final volume 163 ml ngs 2 ml 6 ml 30 ml Binalng Enh ncer final volume 10 ml final volume 30 ml final volume 150 ml 15 ml 18 ml 2x 45 ml Mae BUMer ready to use final volume 60 ml final volume 2 x 150 Elution Buffer 2ml 2x2ml 15 ml Spin Filter 10 50 5 x 50 2 0 ml Receiver Tubes 10 50 5 x 50 1 5 ml Receiver Tubes 10 50 5 x 50 Manual 1 1 1 Initial steps add 42 ml 96 100 add 105 ml 96 100 ethanol to the bottle ethanol to each bottle Wash Buffer Wash Buffer add 8 ml 99 7 add 24 ml 99 7 add 120 ml 99 7 Isopropanol to the Isopropanol to the Isopropanol to the Binding Enhancer Binding Enhancer Binding Enhancer add 7 ml 99 7 add 20 ml 99 7 add 100 ml 99 7 Isopropanol to the Isopropanol to the Isopropanol to the Binding Buffer Bindi
11. ATEC Molecular s ISO 9001 2000 and ISO EN 13485 certified Quality Management System the performance of all components of the MSB Spin PCRapace Invisorb Spin DNA Extraction Kit and the Invisorb Fragment CleanUp 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 MSB Spin PCRapace Invisorb Spin DNA Extraction Kit and the Invisorb Fragment CleanUp 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 PCR product purification DNA Fragment Cleanup 0515 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
12. Avoid microbial contamination of the kit reagents o 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 personnel trained in vitro diagnostic laboratory practice 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 siratec com Microcentrifuge 2 11 100 x g 11 000 rpm Ethanol 96 100 Thermoshaker Pipettes and filter tips Scalpel 120 1 5 ml and 2 0 ml reaction rubes Isopropanol 000000 0 The MSB Spin PCRapace Invisorb Spin DNA Extraction Kit and the Invisorb Fragment CleanUp are validated with 2 Propanol Rotipuran gt 99 7 p a ACS ISO Order no 6752 from Carl Roth Possible suppliers for lsopropanol Carl Roth 2 Propanol Applichem Sigma Rotipuran gt 99 7 p a ACS ISO 2 Propanol f r die Molekularbiologie 2 Propanol Ordering No 6752 Ordering No A3928 Ordering No 59304 1L F 12 12 PCR product purification DNA Fragment Cleanup 0515 Principle and procedure of the MSB Spin PCRapace The MSB Spin PCRapace procedure comprises the following steps o selective binding of DNA fragments to the surface of the DNA Binding Spin Filter o elimination of contaminants
13. Buffer or ddH20 directly onto the center of the Spin Filter H incubate for 1 minute at room temperature centrifuge for 1 minute at 11 000 x g 11 000 rpm DNA in the eluate is now ready to use 14 PCR product purification DNA Fragment Cleanup 0515 Principle and procedure of the Invisorb Spin DNA Extraction Kit The Invisorb Spin DNA Extraction Kit procedure comprises the following steps excise of DNA fragments from the agarose gel with a sharp scalpel gel removal and binding of DNA fragments on the membrane of the spin column selective binding of DNA fragments to the surface of the DNA Binding Spin Filter elimination of contaminants and ethanol elution of highly pure DNA fragment 00000 Sampling and storage of starting material Best results are obtained using freshly prepared DNA slices from gel Do not expose the gel with the DNA fragment to UV light for a long time Reduce cutting time under UV light toa minimum It is damaging to the DNA The samples can be stored for some weeks at 4 C Excise of DNA fragments For best results the pieces of gel should be small as possible Use low melting and standard gels with TAE or TBE buffer systems Gel removal and binding of DNA fragments Gel Solubilizer S in the Invisorb Spin DNA Extraction Kit solubilizes the agarose gel slice under higher temperatures Together with Binding Enhancer and Binding buffer it provides the appropriate condition for the bindin
14. DNA fragments after gel purification for Invisorb Spin DNA Extraction Kit and Invisorb Fragment CleanUp Transfer 30 ul of the extracted PCR product into a 1 5 ml reaction tube Add 2 Units Standard Taq DNA Polymerase no proofreading activity article number 302030XX 3 5 ul Taq Buffer 0 6 ul dNTP s 10 mM each MgCl The final concentration in the mixture must be 1 5 mM Incubate for 15 min at 72 C under continuous shaking in a thermo mixer After this treatment the repaired fragment can be used in cloning ligation experiments 23 PCR product purification DNA Fragment Cleanup 0515 Troubleshooting of the MSB Spin PCRapace and Invisorb Fragment CleanUp Problem Cause Comments and suggestions low recovery poor elution of DNA problems with mineral oil add the elution buffer directly onto the centre of the Spin Filter even if a small elution volume is used apply the correct centrifugation steps Take a higher volume of Binding Buffer Wash once with Binding Buffer Troubleshooting of the Invisorb Spin DNA Extraction Kit and Invisorb Fragment CleanUp Problem Cause Comments and suggestions low recovery problems with down stream application e g ligation incorrect Wash Buffer or no ethanol added poor elution of DNA TBE buffered gels are used ineffective solubilization of the agarose gel slice no Binding Enhancer added contamination with salt
15. R product can be used in subsequent downstream applications sequencing cloning and labeling experiments hybridization transcription digestion with restriction enzymes 00000 Product use limitation The kit works not suitable with PCR products smaller than 70 bp single stranded DNA may diverge PCR product purification DNA Fragment Cleanup 0515 Product characteristics of the Invisorb Spin DNA Extraction Kit Starting material Size of DNA fragments 80 bp 30 kb Time for preparation up to 300 mg of gel slices of from TAE or 70 90 depends on fragment approx 20 min TBE buffered systems length and kind of agarose gel The Invisorb Spin DNA Extraction Kit provides a convenient tool for fast and efficient extraction and purification of DNA fragments directly from TAE and TBE agarose gels slices in less than 20 min in the comfortable spin filter format The purification procedure removes agarose ethidium bromide and other impurities from the DNA sample After gel solubilization DNA fragments from 80 bp up to 30 kb are bound directly onto the membrane of a spin filter column All impurities are removed very efficiently by a washing step The DNA is eluted in as little as 20 ul low salt buffer Invisorb Spin DNA Extraction Kit Binding capacity for fragment size of 200 500 bp up to 10 ug Binding capacity for fragment size of 20 000 kb up to 100 ug Elution volume minimum 20 ul The handling and the composit
16. TS TRAINED IN MOLECULAR BIOLOGICAL TECHNIQUES It is designed to be used with any downstream application employing enzymatic amplification or other enzymatic modifications of RNA 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 kits are developed designed and sold for research purposes only PCR product purification DNA Fragment Cleanup 0515 Product use limitation For purification the fragments should not contain less than 70 bp The maximum length of primers witch can removed is 40 bp DNA fragments should not be bigger than 30 kb and not smaller than 70 bp The included chemicals are only useable once Differing of starting material or flow trace may lead to inoperability therefore neither a warranty nor guarantee in this case will be given neither implied nor express The user is responsible to validate the performance of the STRATEC Molecular Product for any particular use STRATEC Molecular does not provide for validation of performance characteristics of the Product with respect to specific applications STRATEC Molecular Products may be used e g in clinical diagnostic laboratory systems conditioned upon the complete d
17. Therefore a washing step is not required High concentrated pure DNA fragments are eluted ready to use Advantages o ultra fast and easy two step format only binding and elution o excellent purity without washing o 80 95 rate of recovery Invisorb technology is the first technology for the extraction of highly purified nucleic acids using non chaotropic binding conditions Starting from complex biological samples the method allows binding of nucleic acids to nearly all surfaces such as membranes carrier or magnetic particles In combination with specially modified surfaces a selective binding of different nucleic acid targets can be realized Binding of nucleic acids under non chaotropic salt conditions leads to o high binding efficiency o reproducible high yields of ready to use DNA simplified protocols reliable time saving and easy handling steps o guaranteed freedom of operation for all fields of applications o improved quality assurance in the customers laboratories PCR product purification DNA Fragment Cleanup 0515 Product characteristics of the MSB Spin PCRapace Starting material Yield Time for preparation up to 100 ul reaction volume like PCR reaction 80 95 depends approx 5 min mixture on fragment length up to 100 ul restriction digestion mixture up to 100 ul ligation mixture up to 100 ul cDNA synthesis mixture up to 100 ul cycle sequencing reaction mixture The MSB Spin PCRapace provides
18. Up up to 200 ul 60 85 DNA fragments and PCR eS products see ordering information page 28 Agarose gels TAE TBE Invisorb Spin DNA Extraction up to 300 mg 60 90 Invisorb Fragment CleanUp gel slices PCR product purification DNA Fragment Cleanup 0515 Advantages convenient and fast sample processing the most efficient removal of contaminants high recovery rate of PCR products or DNA fragments broad range of fragment sizes 80 bp 30 kb can be purified 0000 The purification procedure has been optimized to remove salts enzymes nucleotides mineral oil agarose ethidium bromide and other impurities from DNA samples Specialized binding buffers promote selective adsorption of DNA fragments and PCR products The pure DNA is eluted in a small volume of buffer or water ready to use for any subsequent application The innovative MSB technology is characterized on the following pages MSB technology represents the fastest technology for purification of DNA fragments with high recovery rates This development opens new possibilities for simplifying the purification procedure and to considerably reduce handling steps and processing time The MSB kits have been designed for extremely efficient purification and or concentration of PCR products or of DNA fragments from enzymatic reaction mixtures with only two steps The DNA fragments bind to the membrane at minimal concentrations of non chaotropic salts
19. ate for 1 minute at room temperature Centrifugation for 1 minute at 11 000 x g 11 000 rpm Important Notes 1 If the PCR mixture contains mineral oil we recommend the addition of 500 ul of Binding Buffer independent of the starting volume It is also possible to wash the bound PCR fragment once with 500 ul of Binding Buffer 2 To increase the final DNA yield we recommend an extended incubation time with Elution Buffer up to 5 minutes which will lead to a slightly higher final yield 3 For concentration of PCR fragments it is possible to elute with lower volume of Elution Buffer than the volume of the starting PCR mixture The minimum volume is 10 ul 4 For ligation mixtures please note that ligation reactions give very often non wanted side products These also are purified and enriched You can order additional Binding Buffer Cat No 10202220 for this application by phone 49 0 30 948928910 01 20 PCR product purification DNA Fragment Cleanup 0515 Protocol 2 Removal of DyeDeoxy terminators from DNA cycle sequencing reactions of PCR products and Plasmids after use ABI Prism terminator Kits Please read the instructions carefully and conduct the prepared procedure for MSB Spin PCRapace and Invisorb Fragment CleanUp Note Before starting with the purification procedure please place a Spin Filter into a 2 0 ml Receiver Tube Attention Please be aware that you have to prepare the Binding Buff
20. er see instruction page 1 Binding of the fluorescent labeled DNA Add 500 ul Binding Buffer to the completed cycle sequencing reaction 20 100 ul and mix very well by pipetting or vortexing Transfer the sample completely onto a Spin Filter and centrifuge for 4 min at maximum speed Attention Optional Step 1 if you need primer close by sequence than perform step one like described below This step may help if sequence has to be readable close up to the starting Oligonucleotide but may reduce purity in some reactions Dye Blubs may appear Also sequence reactions in this region depending on the sequence sometimes show a bad performance Add 500 ul Binding Buffer to the completed cycle sequencing reaction 20 100 ul Add 150 ul of Isopropanol to the mixture and mix very well by pipetting or vortexing Transfer the sample completely onto a Spin Filter and centrifuge for 4 min at maximum speed In case of removal of blobs coming from contamination by primer dimers the addition of Isopropanol is not helpful and should be omitted 2 Elution of the fluorescent labeled DNA Place the Spin Filter into a new 1 5 ml Receiver Tube Add at least 10 ul Elution Buffer or ddH 0 directly onto the center of the Spin Filter Incubate for 1 minute at room temperature Centrifuge for 1 minute at 11 000 x g 11 000 rpm Discard the Spin Filter and proceed with the ABI sample loading 21 PCR product purification DNA Fragment Cleanup
21. es of from TAE or TBE agarose 70 90 depends on fragment approx 20 min gels length and kind of agarose gel The Invisorb Fragment CleanUp provides ultra fast purification and concentration of up to 100 ul PCR products from 80 bp up to 30 kb and of other enzymatic reaction mixtures or of linearized pDNA as well as for the purification of DNA fragments from agarose gels For PCR cleanup the MSB procedure is offered no commonly used washing steps are necessary Finally the DNA fragments will be eluted with low salt buffer or H O For purification of DNA fragments from agarose gels the Invisorb procedure is offered The DNA fragments are bound directly onto the surface of a spin filter column after gel solubilization The DNA fragments will be eluted in a low salt buffer after washing The extraction protocol as well as all buffers is optimized to provide high yield and purity of the recovered DNA fragment The hands on time necessary for the whole procedure is reduced to a minimum The purification process will be ready in 5 20 minutes The purified DNA fragments are ready to use in various downstream applications such as digestion with restriction enzymes hybridization labelling cloning sequencing In vitro Transcription ligation and transformation DNA sequencing amplification microinjection 000000 0 Product use limitation It is not suitable for extraction of circular plasmids because as a result of their ve
22. g of DNA to the silica membrane under high concentrations of salt Removal of contaminants and of ethanol The DNA fragments bind to the membrane contaminants and salts are washed away by the ethanol containing Wash Buffer Any remaining Wash Buffer which may interfere with subsequent enzymatic reactions is removed by additional centrifugation Elution of DNA fragments The efficiency of elution strongly depends on the concentration of salt and on the pH of the elution buffer Unlike to absorption elution is most efficient under basic conditions and low concentrations of salt High concentrated pure DNA fragments are eluted with a minimum of 10 ul of Tris buffer or water Maximum elution efficiency is achieved between pH 7 0 and 8 5 therefore when using water to elute be certain that the pH is within this range DNA must also be stored at 20 C when eluted with water as it is possible that DNA may degrade without a buffering agent DNA yield and concentration DNA yield is dependent on the following three factors the volume of elution buffer how the buffer is applied to the column and the incubation time of the buffer on the column Between 100 and 200 ul of the elution buffer completely covers the Invisorb membrane ensuring maximum yield even when not applied directly to the center of the membrane Elution with up to 50 ul requires the buffer to be added directly to the center of the membrane and an additional 1 2 minute incubat
23. gment purification and concentration MSB technology Invisorb technology Product characteristics of the MSB Spin PCRapace Product characteristics of the Invisorb Spin DNA Extraction Product characteristics of the Invisorb Fragment CleanUp Important notes Important points before starting a protocol Equipment and reagents to be supplied by user Principle and procedure of the MSB Spin PCRapace Preparing reagents and buffers of the MSB Spin PCRapace Scheme MSB Spin PCRapace Principle and procedure of the Invisorb Spin DNA Extraction Preparing reagents and buffers of the Invisorb Spin DNA Extraction Scheme Invisorb Spin DNA Extraction Principle and procedure of the Invisorb Fragment CleanUp Preparing reagents and buffers of the Invisorb Fragment CleanUp Scheme Invisorb Fragment CleanUp Protocol 1 Purification and concentration of DNA fragments from enzymatic reactions like PCR products from PCR reactions cDNA synthesis enzyme restriction digestions Protocol 2 Removal of DyeDeoxy TM terminators from DNA cycle sequencing reactions of PCR products and Plasmids after use ABI Prism TM terminator Kits Special Protocol 3 Purification and concentration of PCR products from 200 ul PCR reactions Protocol 4 Extraction of a DNA fragment from an agarose gel slice Protocol 5 Instruction for repair of A overhangs in DNA fragments after gel purification Troubleshooting Ordering information oO OO N NOD DA oO fF FP WO
24. hnologies Using MSB technology the kit is the ideal tool for ultra fast purification of DNA fragments 80 bp 30 kb after PCR and other enzymatic reactions for the efficient concentration of DNA fragments as well as for the reliable removal of Dye terminators from DNA cycle sequencing reactions The recovery of PCR product is 80 95 and a washing step is not required Using the Invisorb technology the kit is the ideal tool for the efficient extraction of DNA fragments from TAE or TBE agarose gels Trademarks Invisorb MSB Eppendorf Registered marks trademarks etc used in this document even when not specifically marked as such are not to be considered unprotected by law The Invisorb technology is covered by patents and patent applications US 6 110363 US 6 043 354 US 6 037 465 EP 0880535 WO 9728171 WO 9534569 EP 0765335 DE 19506887 DE 10041825 2 WO 0034463 MSB and Invisorb are registered trademarks of STRATEC Biomedical AG The PCR process is covered by US Patents 4 683 195 and 4 683 202 and foreign equivalents owned by Hoffmann La Roche AG 2015 STRATEC Molecular all rights reserved PCR product purification DNA Fragment Cleanup 0515 Contents Kit content of the MSB Spin PCRapace Kit content of the Invisorb Spin DNA Extraction Kit content of the Invisorb Fragment CleanUp Symbols Storage Quality control Safety information Intended use Product use limitation PCR product amp DNA fra
25. iagnostic 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 PCR product amp DNA fragment purification and concentration This manual characterizes innovative kits for membrane adsorption based purification of PCR products or DNA fragments from enzymatic reaction mixtures and of DNA fragments from agarose gels using the high performance MSB or the well established Invisorb technology Different kits were developed and adapted to suit different DNA fragment purification needs Sample kkk Sample Source Recommended Kit Volume Recovery PCR products MSB Spin PCRapace up to 100 ul 80 95 ligation reaction mixture Invisorb Fragment CleanUp enzyme digestion mixture cDNA synthesis mixture cycle sequencing reaction mixture Sample kkk Sample Source Recommended Kit Volume Recovery Strong contaminated DNA Invisorb DNA Clean
26. in DNA Extraction Kit Please read the protocols carefully prior to the start of the preparation procedure Transfer gel slices max 300 mg into a 1 5 ml or 2 0 ml microcentrifuge tube not provided to gel slices up to 150 mg add 500 ul Gel Solubilizer S to gel slices gt 150 mg add 1 ml of Gel Solubilizer S and incubate at 50 C for 10 minutes until the gel is completely solubilized add 250 ul Binding Enhancer follow preparing instructions to a 500 ul reaction volume add 500 ul Binding Enhancer follow preparing instructions to a 1 ml reaction volume and mix by pipetting up and down for 2 3 times load app 800 ul of the sample onto the Spin Filter centrifuge at 11 000 x g 11 000 rpm for 2 min discard the filtrate for reaction volumes gt 800 ul reload the residual volume and repeat the centrifugation step add 500 ul Wash Buffer to the Spin Filter centrifuge for 1 min at 11 000 x g 11 000 rpm discard the filtrate repeat the washing step once again centrifuge for 1 min at 11 000 x g 11 000 rpm discard the filtrate remove the residual ethanol by centrifugation for 4 min at maximum speed transfer the Spin Filter into a new 1 5 ml Receiver Tube add at least 20 ul Elution Buffer directly onto the center of the Spin Filter incubate at room temperature for 5 min centrifuge for 1 minute at 11 000 x g 11 000 rpm DNA is now ready to use ST KA NT 17 PCR product purification DNA Fragment C
27. ion is required for maximum yield if elution is performed with the minimum recommended volume of 10 ul 15 PCR product purification DNA Fragment Cleanup 0515 DNA fragment purification from agarose gel o Gel removal and binding of DNA fragments on the membrane of the spin column o Removal of contaminants and elimination of ethanol o Elution of DNA fragments Gel removal and binding of DNA fragments Gel Solubilizer S in the Invisorb Fragment CleanUp Kit solubilizes the agarose gel slice and together with the binding enhancer and binding buffer provides the appropriate condition for the binding of DNA to the silica membrane under high concentrations of salt Removal of Contaminants The DNA fragments bind to the membrane at minimal concentrations of non chaotropic salts Therefore a washing step is not required Unwanted primers and impurities such as salts enzymes unincorporated nucleotides agarose dyes ethidium bromide oils and detergents do not bind to the silica membrane instead they flow through the column Salts are washed away by the ethanol containing Wash Buffer PE Any remaining Wash Buffer which may interfere with subsequent enzymatic reactions is removed by additional centrifugation Elution of DNA fragments The efficiency of elution strongly depends on the concentration of salt and on the pH of the elution buffer Unlike absorption elution is most efficient under basic conditions and low concentrations of salt
28. ion of the Invisorb Spin DNA Extraction Kit is optimized to guarantee high recovery and purity of the DNA fragments The purified DNA fragments are ready to use for various downstream applications o digestion with restriction enzymes o hybridization labeling cloning o sequencing o in vitro Transcription Product use limitation It is not suitable for purification of circular plasmids because as a result of their very different configurations they were detected in the gel in many positions Furthermore purification of linearized pDNA is possible Important Notes If processing PCR with Taq Polymerase possibly the a overhangs can become lost during the extraction It is recommended to perform reparation of a overhangs subsequent to the extraction if they are needed in the following downstream application see page 26 10 PCR product purification DNA Fragment Cleanup 0515 Product characteristics of the Invisorb Fragment CleanUp The Invisorb Fragment CleanUp is a combination of MSB Spin PCRapace and Invisorb Spin DNA Extraction Kit Starting material Yield Time for Size of DNA fragments 80 bp 30 kb preparation up to 100 ul reaction volume like PCR reaction 80 95 depends on approx 5 min mixture fragment length up to 100 ul restriction digestion mixture up to 100 ul ligation mixture up to 100 ul cDNA synthesis mixture up to 100 ul cycle sequencing reaction mixture up to 300 mg of gel slic
29. leanup 0515 Principle and procedure of the Invisorb Fragment CleanUp The Invisorb Fragment CleanUp Kit combines the convenience of spin column technology with the selective bonding properties of a uniquely designed silica membrane The Invisorb columns are designed to give high end concentrations of purified DNA fragments for subsequent reactions Special buffers provided with each kit are optimized for efficient recovery of DNA and removal of contaminants in each specific application o DNA fragment purification from agarose gel o Cleanup of PCR mixture or of enzymatic reaction mixtures o Two handling options spin columns can be processed in a micro centrifuge or on an Invisorb 96 Vacuum Manifold During DNA cleanup using the Invisorb Fragment CleanUp Kit all enzymes are removed independent of size and secondary structure Protein Molecular weight per enzyme subunit kDa DNA Polymerase 109 Klenow fragment 62 Calf intestinal alkaline phosphatase CIP 69 T4 DNA ligase 55 T4 Polynucleotide kinase 35 Terminal transferase 32 DNase 31 No additional and commonly used washing and drying steps are necessary Finally the DNA fragments will be eluted with low salt buffer or H2O For purification of DNA fragments from agarose gels the DNA fragments from 80 bp up to 30 kb will be bound directly onto the surface of a spin filter column after gel solubilization The DNA fragments will be
30. ng Buffer Binding Buffer Symbols ase Manufacturer Lot number 3 I f Expiry date KE sil l 1 I O Do not reuse Catalogue number Consult operating instructions Temperature limitation PCR product purification DNA Fragment Cleanup 0515 Storage All buffers of the MSB Spin PCRapace Invisorb Spin DNA Extraction Kit and the Invisorb Fragment CleanUp should be stored well sealed and dry at room temperature and are stable for at least 12 months under these conditions Before every use make sure that all components are at room temperature If there are any precipitates within the provided solutions dissolve these precipitates by warming up carefully Room temperature RT is defined as range from 15 30 C Quality control and product warranty STRATEC Molecular warrants the correct function of the MSB Spin PCRapace Invisorb Spin DNA Extraction Kit and the Invisorb Fragment CleanUp 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 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 STR
31. p 50 purifications 1020300200 Invisorb Fragment CleanUp 250 purifications 1020300300 Single components Wash Buffer add 42 ml ethanol 18 ml 1020303000 Elution Buffer 15ml 1020304000 Gel Solubilizer S 30 ml 1020308000 Related products Invisorb DNA CleanUp 50 purifications 1020400200 Invisorb DNA CleanUp 250 purifications 1020400300 Possible suppliers for Isopropanol Carl Roth Applichem Sigma 2 Propanol 2 Propanol f r die Molekularbiologie 2 Propanol Rotipuran gt 99 7 p a ACS ISO Ordering No 6752 Ordering No A3928 Ordering No 59304 1L F 25 PCR product purification DNA Fragment Cleanup 0515 UVTI TTT TTT TTT PPP TTT TTT Sstratecee 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 1A2 1 1 2 2 2 2 1 05 2015
32. ragment Elution of PCR product Elution of DNA fragment 4 ff 4 for detailed information see page 16 and 19 19 PCR product purification DNA Fragment Cleanup 0515 Protocol 1 Purification and concentration of DNA fragments from enzymatic reactions e g PCR products from PCR reactions cDNA synthesis enzyme restriction digestions Please read the instructions carefully and conduct the prepared procedure for MSB Spin PCRapace and Invisorb Fragment CleanUp Note Before starting with the purification procedure please place a Spin Filter into a 2 0 ml Receiver Tube Attention Please be aware that you have to prepare the Binding Buffer see instruction page 14 1 Binding of the PCR or DNA fragments A For PCR mixtures up to 50 ul Add 250 ul Binding Buffer to the PCR sample and mix very well by pipetting or vortexing Transfer the sample completely onto a Spin Filter and centrifuge for 2 min at 11 000 x g 11 000 rpm B For PCR mixture gt 50 ul up to 100 pl Add 500 ul Binding Buffer to the PCR sample and mix very well by pipetting or vortexing Transfer the sample completely onto a Spin Filter and centrifuge for 2 min at 11 000 x g 11 000 rpm Remove the filtrate and centrifuge again for 3 minutes 2 Elution of the PCR or DNA fragments Place the Spin Filter into a new 1 5 ml Receiver Tube Add at least 10 ul Elution Buffer or ddH 0 directly onto the center of the Spin Filter Incub
33. ry different configurations they were detected in the gel in many positions If processing PCR with Taq Polymerase possibly the a overhangs can become lost during the extraction It is recommended to perform reparation of a overhangs subsequent to the extraction if they are needed in the following downstream application see page 26 11 PCR product purification DNA Fragment Cleanup 0515 Important notes Important points before starting a protocol Immediately upon receipt of the Product inspect the Product and its components as well as the package for any apparent damages correct quantities and quality If there are any unconformities you have to notify STRATEC Molecular in writing with immediate effect upon inspection thereof If buffer bottles are damaged contact the STRATEC Molecular Technical Services or your local distributor In case of 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 pipet tips between liquid transfers To avoid cross contamination recommend the use of aerosol barrier pipet tips o All centrifugation steps are carried out at room temperature o When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles o Discard gloves if they become contaminated o Do not mix kit components with components from other kits unless the lot numbers are identical o
34. stratecee molecular User manual MSB Spin PCRapace Invisorb Spin DNA Extraction Kit Invisorb Fragment CleanUp 1020220x00 1020110x00 1020300x00 teal STRATEC Molecular GmbH D 13125 Berlin a for ultra fast purification of DNA fragments after PCR and other enzymatic reactions or and for the extraction of DNA fragments from agarose gels Instruction for the MSB Spin PCRapace With only 5 min of processing time the MSB Spin PCRapace is the fastest system for purification of up to 100 ul PCR products 80 bp 30 kb from dNTP s primers enzymes additives and salts using the unique MSB technology a washing step is not required The kit is useful as well for the cleanup of DNA fragments from salts and enzymes from restrictions digestion ligation and cDNA synthesis mixtures For the efficient concentration of PCR products the MSB Spin PCRapace can be used as well as for the reliable removal of Dye terminators from DNA cycle sequencing reactions The recovery of PCR product is 80 95 Invisorb Spin DNA Extraction The Invisorb Spin DNA Extraction Kit is the ideal tool for extraction of DNA fragments of 80 bp 30 kb from standard or low melt agarose gels in TAE and TBE buffers in high end concentration of DNA Up to 300 mg agarose gel slices can be processed per spin column Invisorb Fragment CleanUp The Invisorb Fragment CleanUp is a combination of different tec
35. the starting volume It is also possible to wash the bound PCR fragment once with 500 ul of Binding Buffer 3 To increase the final DNA yield we recommend an extended incubation time with Elution Buffer up to 5 minutes which will lead to a slightly higher final yield 4 For concentration of PCR fragments it is possible to elute with lower volume of Elution Buffer than the volume of the starting PCR mixture The minimum volume is 10 ul 22 PCR product purification DNA Fragment Cleanup 0515 Protocol 4 Extraction of a DNA fragment from an agarose gel slice Please read the instructions carefully and conduct the prepared procedure for Invisorb Spin DNA Extraction Kit and Invisorb Fragment CleanUp Important TBE gels contain more potentially inhibitors for down stream application than TAE gels So we recommend the use of TAE gels for critical downstream application Before starting with the purification procedure please place a Spin Filter into a 2 0 ml Receiver Tube Attention Please be aware that you have to prepare the Binding Enhancer see instruction page 16 1 Excise the DNA fragment from the agarose gel with a sharp scalpel Minimize the agarose gel slice Check the weight For gel slices up to 150 mg add 500 ul Gel Solubilizer S For gel slices gt 150 mg add 1 ml of Gel Solubilizer S Do not use more than 300 mg gel slice for one Spin Filter Transfer the gel slice into a 1 5 or 2 0 reaction tube 2 Incubate at 5
36. to elute be certain that the pH is within this range DNA must also be stored at 20 C when eluted with water as it is possible that DNA may degrade without a buffering agent Sampling and storage of starting material Best results are obtained using freshly prepared PCR or enzymatic reaction mixtures to prevent DNA digestion The samples can be stored for some weeks at 4 C 13 PCR product purification DNA Fragment Cleanup 0515 Preparing reagents and buffers of the MSB Spin PCRapace 10 preps add 7 ml 99 7 lsopropanol to the Binding Buffer 50 preps add 20 ml 99 7 Isopropanol to the Binding Buffer 250 preps add 100 ml 99 7 Isopropanol to the Binding Buffer 500 preps add 100 ml 99 7 Isopropanol to each Binding Buffer Scheme of MSB Spin PCRapace the preparation procedure up to 50 ul PCR mixtures or enzymatic reaction mixtures add 250 ul Binding Buffer to the PCR sample gt 50 ul up to 100 ul PCR mixtures or enzymatic reaction mixtures 1 Please read the protocols carefully prior to the start of l add 500 ul Binding Buffer follow preparing instructions to the PCR sample Fa mix very well by pipetting up and down or vortexing transfer the sample completely onto the provided Spin Filter centrifuge for 2 min at 11 000 x g 11 000 rpm remove the filtrate and centrifuge again for 3 minutes place the Spin Filter into a new 1 5 ml Receiver Tube add at least 10 ul Elution

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