Invisorb DNA CleanUp User manual
Contents
1. DNA after DNA isolation stratecee molecular User manual Invisorb DNA CleanUp for purification of DNA fragments after PCR reactions amp purification of contaminated 1020400X0 caas STRATEC Molecular GmbH D 13125 Berlin RS Instruction for Invisorb DNA CleanUp The Invisorb DNA CleanUp provides a convenient tool for fast and efficient direct purification of contaminated DNA after DNA isolation as well as for fast and efficient direct purification of PCR products from 80 bp up to 30 kb from amplification reactions Trademarks Invisorb Registered marks trademarks etc used in this document even when not specifically marked as such are not to be considered unprotected by law The Invisorb technology is covered by patents and patent applications US 6 110363 US 6 043 354 US 6 037 465 EP 0880535 WO 9728171 WO 9534569 EP 0765335 DE 19506887 DE 10041825 2 WO 0034463 Invisorb is a registered trademark of STRATEC Biomedical AG The PCR process is covered by US Patents 4 683 195 and 4 683 202 and foreign equivalents owned by Hoffmann La Roche AG 2015 STRATEC Molecular all rights reserved 1 Invisorb DNA CleanUp 0515 Contents Kit contents of Invisorb DNA CleanUp Symbols Storage Quality control Intended use Product use limitation Safety information Product characteristic of Invisorb DNA CleanUp Principle and procedure Important notes Important points before s2. Incubate for 1 min 2 Centrifuge for 1 min at 11 000 x g 11 000 rpm 3 Add 700 ul Wash Buffer to the Spin Filter centrifuge for 1 min at 11 000 x g 11 000 rpm 4 Discard the filtrate Remove the residual ethanol of the Wash Buffer by centrifugation for 4 min at maximum speed 5 Transfer the Spin Filter into a new 1 5 ml Receiver Tube Add at least 30 ul Elution Buffer directly onto the center of the Spin Filter Incubate at room temperature for 3 min 6 Centrifuge for 1 min 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 A longer incubation time with Elution Buffer up to 10 minutes leads also to a slightly higher final yield A prewarming of the Elution Buffer to 70 also increases the recovery Note For purification of PCR fragments from larger amounts of PCR reaction mixtures please call for a special protocol 13 Invisorb DNA CleanUp 0515 Troubleshooting Problem probable cause Comments and suggestions low recovery incorrect Wash Buffer Wash Buffer or Il or no ethanol added poor elution of DNA prepare the Wash Buffer Wash Buffer I or II exactly as described in the manual storage of Wash Buffer with firmly fixed cap add the Elution Buffer directly onto the center of the Spin Filter even if a
3. bottle Wash Buffer I Add 140 ml 96 100 ethanol to the bottle Wash Buffer II Reagents and equipment to be supplied by user Microcentrifuge Measuring cylinder 250 ml Pipette and pipet tips Disposable gloves Reaction tubes 1 5 ml or 2 0 ml Vortexer 96 100 ethanol Isopropanol O O O GEO O The Invisorb DNA CleanUp is validated with 2 Propanol Rotipuran gt 99 7 p a ACS ISO Ordering no 6752 from Carl Roth Possible suppliers for Isopropanol 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 8 Invisorb DNA CleanUp 0515 Scheme of the Invisorb DNA CleanUp Adjustment of binding conditions add 130 ul or 300 ul Buffer P follow preparing instructions to the DNA eluate or PCR mixture incubate for 2 min at RT Binding of the DNA fragments j transfer the complete mixture to the spin column incubate for 2 min at RT centrifuge for 1 min at 11 000 x g 11 000 rpm Washing of the bound fragments and drying the Spin Filter For contaminated DNA add 500 ul Wash Buffer I to the spin column centrifuge for 1 min at 11 000 x g 11 000 rpm add 700 ul Wash Buffer II to the spin column centrifuge for 1 min 11 000 x g 11 000 rpm For PCR cleanup Add 700 ul Wash Buffer to the spin column centrifuge for 1 min at 11 000 x g 11 000 rpm Ethanol removal d
4. completely into a Spin Filter Incubate for 2 min at RT 2 Centrifuge for 1 min at 11 000 x g 11 000 rpm 3 Add 500 ul Wash Buffer I to the Spin Filter centrifuge for 1 min at 11 000 x g 11 000 rpm 4 Add 700 ul Wash Buffer II to the Spin Filter centrifuge for 1 min at 11 000 x g 11 000 rpm 5 Discard the filtrate Remove the residual ethanol of the Wash Buffer II by centrifugation for 4 min at maximum speed 6 Transfer the Spin Filter into a new 1 5 ml Receiver Tube Add at least 30 ul Elution Buffer directly onto the center of the Spin Filter Incubate at room temperature for 3 minutes Centrifuge for 1 min 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 A prewarming of the Elution Buffer to 70 also increases the recovery 10 Invisorb DNA CleanUp 0515 Protocol 2 Purification of contaminated DNA after DNA isolation from up 50 200 ul Please read the instructions carefully and conduct the prepared procedure The following protocol is designed to purify modified and double stranded DNA from using classical procedures and DNA from reaction mixtures like DNA after CTAB purification procedure or DNA isolated from difficult
5. starting materials or DNA from reaction mixtures like the bisulfite method used in methylation analysis resulting in high end concentrations of DNA Fragments ranging from 80 bp to 30 kb are purified from primers nucleotides polymerases and salts using centrifugation driven sample processing Attention Please be aware that you have to prepare the Buffer P see instruction page 8 Before starting with the purification procedure please place a Spin Filter into a 2 0 ml Receiver Tube 1 Mix 300 ul Buffer P with the DNA eluate in a 1 5 ml reaction tube Transfer the suspension completely into a Spin Filter Incubate for 2 min Centrifuge for 1 min at 11 000 x g 11 000 rpm Add 500 ul Wash Buffer I to the Spin Filter centrifuge for 1 min at 11 000 x g 11 000 rpm Add 700 ul Wash Buffer II to the Spin Filter centrifuge for 1 min at 11 000 x g 11 000 rpm Mem e M Discard the filtrate Remove the residual ethanol of the Wash Buffer Il by centrifugation for 4 min at maximum speed 6 Transfer the Spin Filter into a new 1 5 ml Receiver Tube Add at least 30 ul Elution Buffer directly onto the center of the Spin Filter Incubate at room temperature for 3 min 7 Centrifuge for 1 min 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 A long
6. C 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 STRATEC Molecular s ISO 9001 2000 and ISO EN 13485 certified Quality Management System the performance of all components of the Invisorb DNA 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 Invisorb DNA 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 4 Invisorb DNA CleanUp 0515 intended use The Invisorb DNA CleanUp provides a convenient tool for fast and efficient cleanup of strong contaminated DNA DNA fragments and PCR products DNA isolated using classical procedures and DNA from reaction mixtures like the bisulfite method used in methylation analysis DNA purified by the Invisorb DNA CleanUp is ready to use for a broad panel of downstream applications T
7. H032 Contact with acids liberates very toxic gas warning present and easy to do Continue rinsing H 302 31 2 332 412 EUH032 P273 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 Product characteristic of Invisorb DNA CleanUp Starting material Rate of recovery Time for preparation up to 200 ul of DNA eluate 60 85 less than 10 minutes up to 100 ul of amplification reaction depends on fragment length volume up to 100 ul of reaction mixtures The Invisorb DNA CleanUp provides a convenient tool for fast and efficient cleanup of strong contaminated DNA DNA fragments and PCR products 80 bp up to 30 kb DNA isolated using classical procedures and DNA from reaction mixtures like the bisulfite method used in methylation analysis The optimized Binding Buffer adjust the condition and DNA or DNA fragments will be bound directly onto the surface of a spin filter column during contaminats will be passed through during washing step s The buffer volumes are balanced to minimize pipetting steps The Buffer P is added directly to the sample adjusting the binding condition for DNA or DNA fragments and the mixture is applied to the spin filter column The DNA fragments are bound directly onto the membrane of a spin filter column After washing to remove contaminants the DNA or DNA fragments
8. HE PRODUCT IS INTENDED FOR USE BY PROFESSIONALS 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 Product use limitation The Kit is neither for isolation and purification of pDNA nor for isolation and purification of RNA 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 diagnostic system of the laboratory the laboratory has been validated pursuant to CLIA 88 regulations in the U S or equivalent
9. are eluted in a low salt buffer or ddH O The purification protocol as well as all buffers are 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 purified DNA fragments are ready to use in various downstream application such as Digestion with restriction enzymes Hybridization Labeling Cloning Sequencing In vitro Transcription O O O O 0O 0 The PCR method is covered by U S Patents 4 683 195 and 4 683 202 owned by Hoffmann LaRoche Inc The purchase of the Invisorb DNA CleanUp cannot be construed as an authorization or implicit license to practice PCR under any patents held by Hoffmann LaRoche Inc 6 Invisorb DNA CleanUp 0515 Principle and procedure The Invisorb DNA CleanUp procedure comprises following steps 1 adjustment of binding conditions 2 removal of contaminants and elimination of ethanol 3 elution of DNA All steps are performed without use of phenol chloroform CsCl ethidium bromide and without alcohol precipitation This manual contains four protocols Adjustment of binding conditions The Buffer P is simply added directly to the sample adjusting the binding condition for DNA or DNA fragments Binding of DNA or DNA fragments The mixture is applied to an Invisorb Spin column and the DNA or PCR products is adsorbed onto membrane while the contaminating RNA proteins metabolitesm PCR inhibi
10. by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times add 36 ml 96 100 ethanol to the bottle Wash Buffer add 30 ml 96 100 ethanol to the bottle Wash Buffer I add 42 ml 96 100 ethanol to the bottle Wash Buffer II Isopropanol to the Buffer P Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times add 120 ml 96 100 ethanol to each bottle Wash Buffer add 80 ml 96 100 ethanol to the bottle Wash Buffer add 140 ml 96 100 ethanol to the bottle Wash Buffer II Invisorb DNA CleanUp 0515 Symbols eae Manufacturer Lot number r o mij Catalogue number Expiry date Consult operating instructions Temperature limitation BPH Do not reuse Storage The Invisorb DNA CleanUp should be stored dry at room temperature and is stable for at least 12 months under these conditions Make sure that all components have room temperature If there should be any precipitates in the reagents provided dissolve them by careful warming up to 37 C Room temperature RT is defined as range from 15 30 C Quality control and product warranty STRATEC Molecular warrants the correct function of the Invisorb DNA 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 STRATE
11. ctly onto the center of the Spin Filter Incubate at room temperature for 3 minutes Centrifuge for 1 min 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 A prewarming of the Elution Buffer to 70 also increases the recovery 12 Invisorb DNA CleanUp 0515 Protocol 4 Purification of PCR products from PCR reaction mixtures from 50 ul 200 ul Please read the instructions carefully and conduct the prepared procedure The following protocol is designed to purify double stranded DNA fragments from PCR reactions and other enzymatic reactions e g restriction digestion resulting in high end concentrations of DNA Fragments ranging from 80 bp to 30 kb are purified from enzymes primers nucleotides polymerases and salts using the Invisorb Spin columns in a microcentrifuge Attention Please be aware that you have to prepare the Buffer P see instruction page 8 Before starting with the purification procedure please place a Spin Filter into a 2 0 ml Receiver Tube 1 Mix 300 ul Buffer P with the PCR reaction mixture in a 1 5 ml reaction tube mineral oil overlaying does not disturb Transfer the suspension completely into a Spin Filter
12. er incubation time with Elution Buffer up to 10 minutes leads also to a slightly higher final yield A prewarming of the Elution Buffer to 70 also increases the recovery 11 Invisorb DNA CleanUp 0515 Protocol 3 Purification of PCR products from PCR reaction mixes up to 50 ul Please read the instructions carefully and conduct the prepared procedure The following protocol is designed to purify double stranded DNA fragments from PCR reactions and other enzymatic reactions e g restriction digestion resulting in high end concentrations of DNA Fragments ranging from 80 bp to 30 kb are purified from enzymes primers nucleotides polymerases and salts using the Invisorb Spin columns in a microcentrifuge Attention Please be aware that you have to prepare the Buffer P see instruction page 8 Before starting with the purification procedure please place a Spin Filter into a 2 0 ml Receiver Tube 1 Add 130 ul Buffer P directly to the PCR reaction tube mineral oil overlaying does not disturb Transfer the suspension completely into a Spin Filter Incubate for 1 min 2 Centrifuge for 1 min at 11 000 x g 11 000 rpm 3 Add 700 ul Wash Buffer to the Spin Filter centrifuge for 1 min at 11 000 x g 11 000 rpm 4 Discard the filtrate Remove the residual ethanol of the Wash Buffer by centrifugation for 4 min at maximum speed 5 Transfer the Spin Filter into a new 1 5 ml Receiver Tube Add at least 30 ul Elution Buffer dire
13. iscard filtrate centrifuge for 4 min at max speed Elution of purified DNA add 30 100 ul Elution Buffer onto the center of the spin column centrifuge for 1 min at 11 000 x g 11 000 rpm 9 Invisorb DNA CleanUp 0515 Instructions The following notes are valid for all protocols Note The centrifugation steps were made with the Centrifuge 5415 D from Eppendorf The indicated rpm amounts are referring to this centrifuge Protocol 1 Purification of contaminated DNA after DNA isolation from up to 50 ul Please read the instructions carefully and conduct the prepared procedure The following protocol is designed to purify modified and double stranded contaminated DNA isolated by using classical procedures like DNA after CTAB purification procedure or DNA isolated from difficult starting materials including PCR inhibitors or DNA from reaction mixtures like bisulfite method used in methylation analysis resulting in high end concentrations of DNA using centrifugation driven sample processing Fragments ranging from 80 bp to 30 kb are purified from primers nucleotides polymerases and salts using centrifugation driven sample processing Attention Please be aware that you have to prepare the Buffer P see instruction page 8 Before starting with the purification procedure please place a Spin Filter into a 2 0 ml Receiver Tube 1 Add 130 ul Buffer P directly to the tube with the DNA eluate Transfer the suspension
14. ng buffer and tap the tube gently on the side Alternatively let the DNA stand in buffer overnight at 2 to 8 C Minimize vortexing of DNA since this can cause shearing Ordering information Product Package Size Catalogue No Invisorb DNA CleanUp 5 preparations 1020400100 Invisorb DNA CleanUp 50 preparations 1020400200 Invisorb DNA CleanUp 250 preparations 1020400300 14 Invisorb DNA CleanUp 0515 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 1A3p 05 2015
15. s 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 Safety information When and while working with chemicals always wear a suitable lab coat disposable gloves and protective goggles Avoid skin contact Adhere to the legal requirements for working with biological material For more information please consult the appropriate material safety data sheets MSDS These are available online in convenient and compact PDF format at www stratec com for each STRATEC Molecular Product and its components If buffer bottles are damaged or leaking WEAR GLOVES AND PROTECTIVE GOGGLES when discarding the bottles in order to avoid any injuries European Community risk and safety phrases for the components of the Invisorb DNA CleanUp to which they apply are listed below as follows 5 Invisorb DNA CleanUp 0515 Wash Buffer I H302 Harmful if swallowed H312 Harmful in contact with skin H332 Harmful if inhaled H412 Harmful to aquatic life with long lasting effects EU
16. small elution volume is used problems with down stream application e g ligation contamination with salt components washing of the Spin Filters as described in the manual Prolong the incubation time with Wash Buffers to 5 i if ion contamination of the final DNA with MINUtes before centrifugation ethanol keep the given centrifugation time extend it if necessary test the smell Appendix General notes on handling DNA Nature of DNA The length and delicate physical nature of DNA require 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 DNA is necessary to ensure it will function well in various downstream applications Damaged DNA could perform poorly in applications such as Southern blotting and long template PCR Storage of DNA Store DNA and other small circular DNAs at 2 to 8 C Storing pDNA at 15 to 25 C can cause shearing of DNA particularly if the DNA is exposed to repeated freeze thaw cycles Drying dissolving and pipetting DNA Avoid overdrying 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 DNA and other small circular DNAs can be vacuum dried To help dissolve the DNA carefully invert the tubes several times after addi
17. t 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 use kit components from other kits with the kit you are currently using unless the lot numbers are identical o Avoid microbial contamination of the kit reagents 7 Invisorb DNA CleanUp 0515 Preparing reagents and buffers 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 at our webpage www stratec com 1 Label the needed amount of 2 0 ml Receiver Tubes 2 Place spin filters into labeled 2 0 ml Receiver Tubes 3 Label the needed amount of 1 5 ml Receiver Tubes 5 DNA extractions Wash Buffers are ready to use 50 DNA extractions Add 10 ml 99 7 Isopropanol to the Buffer P Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times Add 36 ml 96 100 ethanol to the bottle Wash Buffer Add 30 ml 96 100 ethanol to the bottle Wash Buffer I Add 42 ml 96 100 ethanol to the bottle Wash Buffer II 250 DNA extractions Add 40 ml 99 7 Isopropanol to the Buffer P Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times Add 120 ml 96 100 ethanol to each bottle Wash Buffer Add 80 ml 96 100 ethanol to the
18. tarting a protocol Preparing reagents and buffers Reagents and equipment to be supplied by user Scheme Instructions ON N N EO ma GG Ma it fk A Q 10 Protocol 1 Purification of contaminated DNA after DNA isolation from up to 50 ul 10 Protocol 2 Purification of contaminated DNA after DNA isolation up to 50 200 ul 11 Protocol 3 Purification of PCR products from PCR reaction mixes up to 50 ul 12 Protocol 4 Purification of PCR products from PCR reaction mixes up to 50 200ul 13 Troubleshooting Appendix General notes on handling DNA Ordering information 14 14 14 14 Invisorb DNA CleanUp 0515 Kit contents of Invisorb DNA CleanUp Store all kit components at room temperature 5 purifications 50 purifications 250 purifications Catalog No Buffer P Wash Buffer 1020400100 2 x1 ml 5 ml ready to use 1020400200 10 ml final volume 20 ml 24 ml final volume 60 ml 1020400300 40 ml final volume 80 ml 80 ml final volume 200 ml Wash Buffer I 15 ml ready to use 30 ml final volume 60 ml 80 ml final volume 160 ml Wash Buffer Il 15 ml ready to use 18 ml final volume 60 ml 60 ml final volume 200 ml Elution Buffer 2ml 15 ml 30 ml Spin Filter 5 50 5 x 50 i Receiver 5 50 5 x 50 yi Receiver 5 50 5 x50 Manual 1 1 1 Initial steps add 10 ml 99 7 add 40 ml 99 7 Isopropanol to the Buffer P Mix
19. tors or nucleotides salts primer polymerase etc remain in the lysate and are drawn through by centrifugal force Removing residual contaminants Contaminations like endonucleases or others are efficiently washed away using the relevant Wash Buffers while the DNA remains bound to the membrane Elution of DNA DNA is eluted from the column using 50 100 ul Elution Buffer Eluting twice each with 30 100 ul leads to slightly recovery of DNA By the use of small elution volumes DNA concentration can be raised Elution volumes should not fall below 30 ul otherwise the yield will be reduced The eluted DNA is ready to use in different downstream applications 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 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 are carried out a
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