Invisorb Spin Swab Kit - STRATEC Biomedical AG
Contents
1. Elution Buffer 2 ml 15 ml 30 ml Spin Filter 5 50 5x50 2 0 ml Receiver 5 50 5 x 50 Tubes 1 5 ml Receiver 5 50 5 x 50 Tubes Sterile swabs 5 50 5x50 Manual 1 1 1 Initial steps The Wash Buffer is ready Add 21 ml 99 796 Add 84 ml 99 796 to use The Binding Buffer A is ready to use Add 250 ul dd H20 to the tube with Proteinase K mix thoroughly until completely dissolving and store at 20 C Incubate the needed amount of Elution Buffer at 65 C Isopropanol to the Binding Buffer A Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times Add 42 ml of 96 100 Ethanol to the bottle Wash Buffer mix thoroughly and keep the bottle always firmly closed Add 1 1 ml dd HO to the tube with Proteinase K mix thoroughly until completely dissolving and store at 20 C Incubate the needed amount of Elution Buffer at 65 C Isopropanol to the Binding Buffer A Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times Add 140 ml of 96 100 Ethanol to each bottle Wash Buffer mix thoroughly and keep the bottle always firmly closed Add 1 1 ml dd HO to each tube with Proteinase K mix thoroughly until completely dissolving and store at 20 C Incubate the needed amount of Elution Buffer at 65 C Invisorb Spin Swab Kit 0515 Symbols OT Lot number Catalogue number2. see page 7 Do not use damaged kit components since their use may lead to poor kit performance o Always change pipette tips between liquid transfers To avoid cross contamination we recommend the use of aerosol barrier pipette tips o All centrifugation steps are carried out at room temperature When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles Discard gloves if they become contaminated Do not combine components of different kits unless the lot numbers are identical Avoid microbial contamination of the kit reagents To minimize the risk of infections from potentially infectious material we recommend working under laminar air flow until the samples are lysed o This kit should only be used by trained personnel o O O 0 0 8 Invisorb Spin Swab Kit 0515 Preparing reagents and buffers for the Invisorb Spin Swab Kit Before starting a run bring all reagents to room temperature Where necessary gently mix and re dissolve any precipitates by incubation at 30 C Swirl gently to avoid foaming Lysis Buffer G Binding Buffer A and Elution Buffer are ready to use Add the needed volume of ddH O see Kit Contents page 3 to the reaction tube with Proteinase K Vortex for 5 sec store diluted Proteinase K at 20 C 1 adjust the thermomixer to 65 C 2 warm up the needed amount of Elution Buffer to 65 C 50 100 ul Elution Buffer are needed per sample label
3. Kit contents of Invisorb Spin Swab Kit 3 Symbols 4 Storage 4 Quality control 4 Intended use 5 Product use limitation 5 Safety information 6 Product characteristic of the Invisorb Spin Swab Kit 7 Principle and procedure 8 Important notes 9 Important points before starting a protocol 9 Preparing reagents and buffers for the Invisorb Spin Swab Kit 9 Reagents and equipment to be supplied by user 10 Important indications 10 Scheme of the Invisorb Spin Swab Kit 11 Protocol 1 DNA Extraction from swab material 12 Protocol 2 DNA Extraction from swab material especially for isolation of bacterial DNA particular gram bacteria or other hard to lyse pathogens 13 Troubleshooting 14 Appendix 15 Ordering information 16 Invisorb Spin Swab Kit 0515 Kit contents of Invisorb Spin Swab Kit Store diluted Proteinase K at 20 C but repeated freezing and thawing will reduced the activity dramatically Dividing the Proteinase K into aliquots and storage at 20 C is recommended 5 DNA preps 50 DNA preps 250 DNA preps Catalogue No 1035120100 1035120200 1035120300 Lysis Buffer G 2x2ml 50 ml 160 ml 2x1 ml 9 ml 36 ml Binding Buffer A ready to use final volume 30 ml final volume 120 ml Proteinase K for 250 ul for 1 1 ml for 5 x 1 1 ml working solution working solution working solution 15 ml 2x 18 ml 2x 60 ml Wash Buffer ready to use final volume 2 x 60 ml final volume 2 x 200 ml
4. dry the swab for at least 2 h after collection or use them fresh prepared Ensure that the person providing the sample has not consumed any food or drink in the 30 min prior to sample collection Best results are obtained if the swab stays in the lysis solution during lysis procedure Use of poor quality starting material influences yield of purified DNA This protocol is recommended for every common swab like e g the following swab types C E P Omni Swab from Whatman cotton swab Superswabs Copan Swab or DRACON tip from Hardwood Products company CellProjects or Hain Diagnostika STRATEC Molecular will be released of its responsibilities if other sample materials than described in the Intended Use are processed or if the sample preparation protocols are changed or modified Lysis with Proteinase K Samples are lysed under anti chaotropic conditions at elevated temperature and continuously shaking Lysis is performed in the presence of Lysis Buffer G and Proteinase K By crushing or grinding the sample under liquid nitrogen the lysis efficiency is dramatically increased and lysis time is reduced Using rodent tails an overnight lysis is possible Unlysed sample parts should be removed before the binding step The PCR process is covered by US Patents 4 683 195 and 4 683 202 and foreign equivalents owned by Hoffmann La Roche AG 7 Invisorb Spin Swab Kit 0515 Binding genomic DNA By adding Binding Buffers A to the lysate optimal bindin
5. H 1 Expiry date U 7 iz Consult operating instructions Temperature limitation Do not reuse E Manufacturer Storage All buffers and kit contents of the Invisorb Spin Swab Kit except dissolved Proteinase K should be stored at room temperature RT and are stable for at least 12 months under these conditions Dissolved Proteinase K must be stored at 20 C Wash Buffer charged with ethanol should be stored at room temperature and should be appropriate sealed If there are any precipitates within the provided solutions dissolve these precipitates by carefully warming up to room temperature 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 Spin Swab Kit for applications as described in this manual Purchaser must determine the suitability of the Product for its particular use Should any Product fail to perform the applications as described in the manual STRATEC Molecular will check the lot and if STRATEC Molecular investigates a problem in the lot STRATEC Molecular will replace the Product free of charge STRATEC Molecular reserves the right to change alter or modify any Product to enhance its performance and design at any time In accordance with STRATEC Molecular s ISO 9001 2000 and ISO EN 13485 certified Quality Management System the performance of all components of the Invisorb Spin Swab Kit h
6. age 9 Important Prewarm the Elution Buffer to 65 e g transfer the needed volume into a reaction tube and place the tube at the appropriate temperature 1 Lysis of the starting material Transfer 600 ul of Lysis Buffer G and 20 yl of Proteinase K into a 1 5 ml Reaction Tube Transfer the swab into the so prepared tube and incubate the sample at 65 C for 15 minutes under continuously shaking e g by using a thermo mixer Important To get maximum yield of DNA it is essential to leave the swab during the complete lysis time into the reaction tube It is possible to cut the shaft of the swab so that you can close the cap of the reaction tube It is also possible to do the lysis step with opened cap The removing of the swab from the reaction tube ahead of time will be lead to a dramatically reduced final yield After lysis time carefully squeeze out the swab on the wall of the tube and discard the swab 2 Realizing optimal binding conditions Add 300 ul of Binding Buffer A to the reaction tube and mix thoroughly Set a Spin Filter into a 2 0 ml Receiver Tube and transfer the suspension onto the Spin Filter Centrifugation for 2 min at 11 000 x g 11 000 rpm Open the Receiver Tube take the Spin Filter from the Receiver Tube and discard the filtrate 3 Washing of the Spin Filter Place the Spin Filter back into the 2 0 ml Receiver Tube and add 700 ul Wash Buffer to the Filter Centrifugation at 11 000 x g 11 000 rpm for 1 min T
7. ake the Filter from the Receiver Tube discard the filtrate and then place the Spin Filter again into the Receiver Tube Repeat the washing step once Discard the filtrate again and put the Spin Filter back into the Receiver Tube Close the Tube and centrifuge for 4 min at maximum speed for complete removal of Ethanol 4 Elution of DNA Place the Spin Filter into a new 1 5 ml Receiver Tube and add 50 100 ul of prewarmed Elution Buffer Incubation for 1 min at room temperature Close the Receiver Tubes and centrifuge for 1 min at 11 000 x g 11 000 rpm Discard the Spin Filter The filtrate contains the pure DNA Note The DNA can also be eluted with a lower volume of Elution Buffer It is also possible to do the elution step two times with equal volumes of Elution Buffer These will lead to slightly increased total yield But pay attention that minimum volume for the elution is 40 ul Note The centrifugation steps were made with the Centrifuge 5415 D from Eppendorf The indicated rpm amounts are refering to this centrifuge 12 Invisorb Spin Swab Kit 0515 Protocol 2 DNA Extraction from swab material especially for isolation of bacterial DNA particular gram bacteria or other hard to lyse pathogens Please read the instructions carefully and conduct the prepared procedure Aitention Please be aware that you have to prepare the Binding Buffer A see instruction page 9 Important Prewarm the Elution Buffer to 65 e g
8. at 20 C but storing at 20 C can cause shearing particularly if the DNA is exposed to repeated freeze thaw cycles Note that the solution in which the nucleic acid is eluted in will affect it s stability during storage Pure water lacks buffering capacity and an acidic pH may lead to acid hydrolysis Tris or Tris EDTA buffer contains sufficient buffering capacity to prevent acid hydrolysis Drying dissolving and pipetting DNA Avoid over drying genomic DNA after ethanol precipitation It is better to let it air dry than to use a vacuum although vacuum drying can be used with caution Avoid vigorous pipetting Pipetting genomic DNA through small tip openings causes shearing or nicking One way to decrease shearing of genomic DNA is to use special tips that have wide openings designed for pipetting genomic DNA DNA yield The amount of purified DNA from the swab samples depends on sample source transport conditions storage and age of the sample 15 Invisorb Spin Swab Kit 0515 Ordering information Product Package Size Catalogue No Invisorb Spin Swab Kit 5 preps 1035120100 Invisorb Spin Swab Kit 50 preps 1035120200 Invisorb Spin Swab Kit 250 preps 1035120300 Single components for the Invisorb Spin Swab Kit Lysis Buffer G 60 ml 1035121100 Binding Buffer A add 21 ml 15 ml 1035122100 Wash Buffer add 42 ml 18 ml 1035123200 Elution Buffer 00 i5ml 1035124000 s Related products PSP SalivaGene DNA Kit 50 pr
9. ave been tested separately against predetermined specifications routinely on lot to lot to ensure consistent product quality If you have any questions or problems regarding any aspects of Invisorb Spin Swab Kit or other STRATEC Molecular products please do not hesitate to contact us A copy of STRATEC Molecular s terms and conditions can be obtained upon request or are presented at the STRATEC Molecular webpage For technical support or further information please contact from Germany 49 0 30 9489 2901 2910 from abroad 49 0 30 9489 2907 or contact your local distributor Invisorb Spin Swab Kit 0515 Intended use The Invisorb Spin Swab Mini Kit is the ideal tools using the Invisorb technology for manual isolation and purification of genomic DNA from f swabs like buccal nasal pharyngeal and vaginal swabs for in vitro diagnostic analysis For reproducible and high yields appropriate sample storage is essential The purified DNA can be used for in vitro diagnostic analysis only THE 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 sh
10. eparations 1035200200 PSP SalivaGene DNA Kit 250 preparations 1035200300 SalivaGene Collection Module II 5 container 1035210600 SalivaGene Collection Module II 50 container 1035210700 SalivaGene Collector 5 pieces 1035210100 SalivaGene Collector 50 pieces 1035210200 SalivaGene Buccal Swab 5 pieces 1035230100 SalivaGene Buccal Swab 50 pieces 1035230200 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 Order no A3928 Order no 59304 1L F Order no 6752 16 Invisorb Spin Swab Kit 0515 stratecee molecular STRATEC Molecular GmbH Robert R ssle Str 10 13125 Berlin Germany Phone 49 30948929 01 Fax 49 30 94 89 29 09 E mail info berlin stratec com www stratec com 1A3g 05 2015
11. form 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 Invisorb Spin Swab Kit 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 discarding the bottles in order to avoid any injuries STRATEC Molecular has not tested the liquid waste generated by the Invisorb Spin Swab Kit procedures for residual infectious materials Contamination of the liquid waste with residual infectious materials is highly unlikely but cannot be excluded completely Therefore liquid waste must be considered infectious and be handled and discarded according to local safety regulations Patient specimens must always be considered as potentially infectious Samples from risk patients must alway
12. g conditions are adjusted Each lysate is then applied to an Invisorb Spin Filter and genomic DNA is adsorbed onto the membrane as the lysate is drawn through by centrifugal force as contaminants pass through Removing residual contaminants Remaining contaminants and enzyme inhibitors are efficiently removed in two efficient wash steps using Wash Buffer while the genomic DNA remains bound to the membrane Elution of pure genomic DNA Genomic DNA ready for use is eluted from the Spin Filter using 50 100 ul Elution Buffer or water Invisorb purified DNA has Azgo A 280 ratios of 1 6 2 0 and absorbance scans show a symmetric peak at 260 nm confirming purify Eluting twice by splitting the elution volume in two parts leads to little increase of DNA yield The usage small elution volumes may raise DNA concentration Elution volumes should be at least 50 ul or 150 ul The eluted DNA is ready for 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
13. ng the complete lysis time into the reaction tube increase lysis time increase centrifugation speed reduce amount of starting material elute the DNA with lower volume of Elution Buffer old material often contains degraded DNA low amount of extracted DNA insufficient lysis incomplete elution insufficient mixing with Binding Buffer A increase lysis time reduce amount of starting material overloading of spin filter reduces yield prolong the incubation time with Elution Buffer to 5 10 min or repeat elution step once again take higher volume of Elution Buffer mix sample with Binding Buffer A by pipetting or by vortexing prior to transfer the sample onto the spin column Invisorb Spin Swab Kit 0515 Appendix General notes on handling DNA Nature of DNA The length and delicate physical nature of DNA requires careful handling to avoid damage due to shearing and enzymatic degradation Other conditions that affect the integrity and stability of DNA include acidic and alkaline environments high temperature and UV irradiation Careful isolation and handling of high molecular weight DNA is necessary to ensure compatibility with various downstream applications Damaged DNA could perform poorly in applications such as genomic Southern blotting long template PCR Storage of DNA A working stock of DNA can be stored at 2 4 C for several weeks For long term storage DNA should be stored
14. nology to provide an extremely fast way to isolate genomic DNA from above named starting material The purified DNA is free of contaminants and enzyme inhibitors and performs reliably in downstream applications such as PCR Purifications require no phenol or chloroform extraction or alcohol precipitation No toxic or hazardous chemicals like chaotropic components are used The kit is designed for simultaneous processing of multiple samples The procedure requires minimal interaction by the user allowing safe handling of potentially infectious samples The procedures are designed to avoid sample to sample cross contamination Purified DNA is eluted in a low salt buffer without EDTA or water Due to the high purity the isolated genomic DNA is ready to use for a broad panel of downstream applications see below or can be stored at 20 C for subsequent use o PCR o HLA Typing o RFLP Analysis o Cloning o Restriction Enzyme Digestion For further information please contact Phone 49 0 30 9489 2901 2910 in Germany and from foreign countries phone 49 0 30 9489 2907 Principle and procedure The Invisorb Spin Swab Kits simple procedure comprises following steps 1 lysis of cells 2 binding the genomic DNA to the membrane of a Spin Filter 3 washing the membrane and elimination of ethanol 4 elution of genomic DNA Sample collection and storage To collect a sample scrape the swab firmly against inside of each cheek 6 times Air
15. ntrifuge Thermomixer for 65 C Measuring cylinder 250 ml Pipette and pipette tips Disposable gloves Reaction tubes 1 5 ml or 2 0 ml dd H2O Vortexer 96 100 Ethanol Isopropanol OOO O E Aa DO 0O0 00 The Invisorb Spin Swab Kit is validated with 2 Propanol Rotipuran gt 99 7 p a ACS ISO Order 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 Order no 6752 Order no A3928 Order no 59304 1L F Important indications 1 Invisorb Spin Filter can also purify low amounts of RNA besides DNA For the elimination of RNA if necessary add 40 ul RNase A 10 mg ml before adding the Binding Buffer A After that vortex shortly and incubate the sample at room temperature for 5 minutes Then go on as described in the protocol 2 The elution can be done by using lower amount of Elution Buffer min 50 ul This may result in a higher DNA concentration 3 Eluting twice with each with 50 ul Elution Buffer is also possible and produces slightly higher yield of DNA 4 Co purification of RNA The kit co purifies DNA and RNA when both are present in the same sample Samples which contain high level of RNA RNA will be co purified RNA may inhibit some downstream enzymatic reactions although it does not affect PCR If RNA free genomic DNA is required RNase A should be added to the sample befo
16. ould be interpreted with regard to other Clinical or laboratory findings To minimize irregularities in diagnostic results adequate controls for downstream applications should be used The kit is in compliance with EU Directive 98 79 EC on in vitro medical devices But it is not for in vitro diagnostic use in countries where the EU Directive 98 79 EC on in vitro medical devices is not recognized Product use limitation The kit is validated neither for the isolation of DNA from stool samples blood tissue fungi or viruses 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 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 per
17. re addition of Binding Buffer A to digest the RNA 10 Invisorb Spin Swab Kit 0515 Scheme of the Invisorb Spin Swab Kit Genomic DNA Please read protocols prior the start of the preparation Pipette 600 ul Lysis Buffer G and 20 ul Proteinase K to the sample 5 10 s mix thoroughly e g vortexing Incubate at 65 C while continuously shaking for 15 min Add 300 ul Binding Buffer A follow preparing instructions mix thoroughly Place a Spin Filter into a 2 0 ml Receiver Tube Transfer the suspension onto the Spin Filter Centrifugation for 2 min at 11 000 x g 11 000 rpm Discard filtrate Add 700 ul Wash Buffer onto Spin Filter Centrifuge for 1 min at 11 000 x g 11 000 rpm Discard filtrate Place the Spin Filter again into the 2 0 ml Receiver Tube Repeat the Washing step Discard filtrate Place Spin Filter again into the 2 0 ml Receiver Tube Centrifuge for 4 min at maximum speed for ethanol removal Place the Spin Filter into a 1 5ml Receiver Tube Add 50 100 ul prewarmed Elution Buffer Incubate at room temperature for 3 min Centrifuge for 1 min at 11 000 x g 11 000 rpm Discard the Spin Filter The eluate contains ready to use DNA 11 Invisorb Spin Swab Kit 0515 Protocol 1 DNA Extraction from swab material Please read the instructions carefully and conduct the prepared procedure Attention Please be aware that you have to prepare the Binding Buffer A see instruction p
18. s be labeled and handled under suitable safety conditions Observe all federal state and local safety and environmental regulations Observe the usual precautions for nucleic acid applications It is essential that all reagents and materials used for DNA isolation are free from DNases European Community risk and safety phrases for the components of the Invisorb Spin Swab Kit to which they apply are listed below as follows Proteinase K OD danger H315 319 334 335 P280 305 351 338 310 405 H319 Causes serious eye irritation H315 Causes skin irritation H334 May cause allergy or asthma symptoms or breathing difficulties if inhaled H335 May cause respiratory irritation P305 P351 P338 IF IN EYES Rinse cautiously with water for several minutes Remove contact lenses if present and easy to do Continue rinsing P280 Wear protective gloves protective clothing eye protection face protection P310 Immediately call a POISON CENTER or doctor physician P405 Store locked up Emergency medical information can be obtained 24 hours a day from infotrac outside of USA 1 352 323 3500 in USA 1 800 535 5053 6 Invisorb Spin Swab Kit 0515 Product characteristic of the Invisorb Spin Swab Kit r Time for buccal nasal up to 6 ug depends of kind of A260 A 280 pharyngeal vaginal swabs starting material 1 7 2 0 The Invisorb Spin Swab Kit uses the well established Invisorb tech
19. stratecee molecular User manual Invisorb Spin Swab Kit For genomic DNA purification from from clinical swab material 1035120x00 MAN cTRATEC Molecular GmbH D 13125 Berlin v p Instruction for Invisorb Spin Swab Kit The Invisorb Spin Swab Kit is the ideal tool using the Invisorb technologies for manual isolation and purification of DNA from swabs like buccal nasal pharyngeal and vaginal swabs for in vitro diagnostic analysis The kit is neither validated for the isolation of DNA from stool samples blood samples fungi plants or viruses nor for purification of RNA CE o Not for in vitro diagnostic use in countries where the EU Directive 98 79 EC on in vitro medical devices is not recognized Compliance with EU Directive 98 79 EC on in vitro medical devices 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 Invisorb Spin Swab Kit 0515 Contents
20. the needed amount of 2 0 ml Receiver Tubes place Spin Filters into labeled 2 0 ml Receiver Tubes label the needed amount of 1 5 ml Receiver Tubes add the needed ddH O see Kit Contents page 3 to the reaction tube with Proteinase K vortex for 5 sec store dissolved Proteinase K at 20 7 add the needed amount of ethanol to the Wash Buffer 9 UY ee 5 DNA extractions Add 250 pl dd H20 to the tube Proteinase K mix thoroughly until completely dissolving and store at 20 C The Wash Buffer and the Binding Buffer A is ready to use 50 DNA extractions Add 21 ml 99 7 Isopropanol to the Binding Buffer A Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times Add 1 1 ml dd H 0 to the tube Proteinase K mix thoroughly until completely dissolving and store at 20 C Add 42 ml of 96 10096 ethanol to the bottle Wash Buffer mix thoroughly and always keep the bottle firmly closed 250 DNA extractions Add 84 ml 99 7 Isopropanol to the Binding Buffer A Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times Add 1 1 ml dd H O to each tube Proteinase K mix thoroughly until completely dissolving and store at 20 C Add 140 ml of 96 100 ethanol to the bottle Wash Buffer mix thoroughly and always keep the bottle firmly closed 9 Invisorb Spin Swab Kit 0515 Reagents and equipment to be supplied by user Microce
21. transfer the needed volume into a reaction tube and place the tube at the appropriate temperature 1 Lysis of the starting material Transfer 600 ul of Lysis Buffer G and 20 yl of Proteinase K into a 1 5 ml Reaction Tube Transfer the swab into the so prepared tube and incubate the sample at 65 C for 15 minutes under continuously shaking e g by using a thermomixer Important To get maximum yield of DNA it is essential to leave the swab during the complete lysis time into the reaction tube It is possible to cut the shaft of the swab so that you can close the cap of the reaction tube It is also possible to do the lysis step with opened cap The removing of the swab from the reaction tube ahead of time will be lead to a dramatically reduced final yield After lysis time carefully squeeze out the swab on the wall of the tube and discard the swab Incubate the sample for additional 30 minutes under continuously shaking at 95 C This step will lead to a thermic disintegration of bacterial cell wall structures 2 Realizing optimal binding conditions Add 300 ul of Binding Buffer A to the reaction tube and mix thoroughly Set a Spin Filter into a 2 0 ml Receiver Tube and transfer the suspension onto the Spin Filter Centrifugation for 2 min at 11 000 x g 11 000 rpm Open the Receiver Tube take the Spin Filter from the Receiver Tube and discard the filtrate 3 Washing of the Spin Filter Place the Spin Filter back into the 2 0 ml Recei
22. ver Tube and add 700 ul Wash Buffer to the Filter Centrifugation at 11 000 x g 11 000 rpm for 1 min Take the Filter from the Receiver Tube discard the filtrate and then place the Spin Filter again into the Receiver Tube Repeat the washing step once Discard the filtrate again and put the Spin Filter back into the Receiver Tube Close the Tube and centrifuge for 4 min at maximum speed for complete removal of Ethanol 4 Elution of DNA Place the Spin Filter into a new 1 5 ml Receiver Tube and add 50 100 ul of prewarmed Elution Buffer Incubation for 1 min at room temperature Close the Receiver Tubes and centrifuge for 1 min at 11 000 x g 11 000 rpm Discard the Spin Filter The filtrate contains the pure DNA Note The DNA can also be eluted with a lower volume of Elution Buffer It is also possible to do the elution step two times with equal volumes of Elution Buffer These will lead to slightly increased total yield But pay attention that minimum volume for the elution is 40 ul Note The centrifugation steps were made with the Centrifuge 5415 D from Eppendorf The indicated rpm amounts are refering to this centrifuge 13 Invisorb Spin Swab Kit 0515 Troubleshooting Problem Cause Comments and suggestions clogged Spin Filter low concentration of extracted DNA Degraded or sheared DNA insufficient lysis and or too much starting material too much Elution Buffer old material incubate the swab duri
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