PSP SalivaGene DNA Kit User manual
Contents
1. 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 perform as described 5 PSP SalivaGene DNA Kit 0615 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 requiremen2. and saliva sample stratecee molecular User manual PSP SalivaGene DNA Kit for purification of total DNA from SalivaGene stabilized clinical swab mouth brushes 1035200X00 oms STRATEC Molecular GmbH D 13125 Berlin A Instruction for PSP SalivaGene Systems PSP SalivaGene Systems are different tools to stabilize and purify genomic and bacterial DNA quantitatively from saliva and swabs e g mouth brushes The PSP SalivaGene DNA Kit in combination with the SalivaGene Buccal Swab the SalivaGene Collection Module II SalivaGene Collector or the SalivaGene Collector Premium SalivaGene Collection Sets is a modular system using a stabilization solution for collection stabilization storage and transportation of swab or saliva material without degradation of DNA for at least 12 month with a very efficient isolation method for high quality DNA The kit has been designed for isolation of genomic mitochondrial and bacterial DNA for in vitro diagnostic analysis The kit is suitable neither for the isolation of DNA from stool samples or from fungi nor for purification of total RNA C Compliance with EU Directive 98 79 EC on in vitro medical devices Not for in vitro diagnostic use in countries where the EU Directive 98 79 EC on in vitro medical devices is not recognized Trademarks Invisorb PSP MSB MSB Registered marks trademarks etc used in this document even when not specifi
3. Tube discard the filtrate and then place the Spin Filter again into the Receiver Tube Repeat the washing step 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 5 Elution of DNA Place the Spin Filter into a new 1 5 ml Receiver Tube and add 50 100 ul of prewarmed Elution Buffer Incubate for 1 min at room temperature Close the Spin Filter 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 30 ul 15 PSP SalivaGene DNA Kit 0615 Protocol II Bacterial DNA extraction from stabilized material by the PSP SalivaGene DNA Kit Please read the instructions carefully and conduct the prepared procedure Important Transfer the needed amount of Elution Buffer into 2 0 ml Receiver Tube not included in the kit and place the tube at 48 Important Bacterial swabs must be taken by medical staff Attention Please be aware that you have to prepare the Binding Buffer A see instruction page 9 1 Protein removal Transfer 500 ul of Sample DNA Stabilizing Solution from the SalivaGene Co
4. approx 2 ml of liquid DNA Stabilizer reagent Limitations of the procedure Do not eat drink smoke or chew gum at least 30 min prior to swab collection Ensure the swab tip does not come into contact with any surface prior to collection Put the Stabilizer tube upright to prevent the liquid inside the tube from spilling Be sure to move the swab over the entire cheek and to moisten it with saliva Open swab package and remove swab without touching the tip Insert swab into the mouth and rub swab tip firmly against the inner cheeks for about 30 seconds on each side Unscrew lid from Stabilizer tube and put it aside for further use Insert swab into the Stabilizer tube Break swab tip at breaking point Discard the broken handle part Screw lid tightly onto the Stabilizer tube again Gently shake tube for 15 sec to mix buccal cells and DNA Stabilizer reagent Label Stabilizer tube with donor name and collection date using provided label 13 PSP SalivaGene DNA Kit 0615 Scheme of the PSP SalivaGene DNA Kit Please read protocols prior the start of the preparation carefully Transfer 500 ul aliquots of the stabilized sample from the SalivaGene Collection Sets to a 1 5 ml Reaction Tube not provided 1 Protein removal Add 20 ul of Proteinase K Incubate for 20 minutes at 48 C under continuously shaking 2 Realizing optimal binding conditions Add 200 yl of Binding Buffer A follow prep
5. on the same day as collection or stored for future processing Long term storage of the sample should be done at 20 C 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 Principle and procedure The PSP SalivaGene DNA Kit procedure comprises following steps o collection of the material using one of the SalivaGene Collection Sets o lysis of cells under non chaotropic conditions at elevated temperature in the Stabilization Buffer SalivaGene DNA Stabilizer protein digestion binding genomic DNA to the membrane of a RTA Spin Filter washing the membrane and elimination of contaminants and ethanol elution of pure genomic DNA O ONO 0 This manual contains 2 protocols according to the different requirements of the starting materials Protein digestion Proteins will be digested in the lysed samples in the presence of Proteinase K Binding genomic DNA By adding Binding Buffer A to the lysate optimal binding conditions will be adjusted Each lysate is then applied to an RTA Spin Filter and genomic DNA is adsorbed to the membrane Removing residual contaminations Contaminants are efficiently washed away using Wash Buffer SL and 70 Ethanol while the total DNA remains bound to the membrane Elution of pure genomic DNA Genomic DNA is eluted from the Spin Filter using 50 100 ul
6. Sample DNA Stabilizing Solution from the SalivaGene Collection Sets into a 1 5 ml reaction tube not provided Add 20 ul of Proteinase K and incubate for 20 min at 48 C under continuously shaking or every 5 minutes flicking the tube Note f you have to archive a swab sample e g for forensic purposes please use only 250 ul of the sample for preparation In this case you add 250 ul of water to the stabilization solution and continue with the protocol as follows If you have to archive a SalivaGene sample e g for forensic purposes just freeze the rest you don t use for preparation The rest of the stabilized sample can be stored at 20 for years 2 Binding of DNA to the RTA Spin Filter Add 200 yl of Binding Buffer A to the 1 5 ml reaction tube mix well Transfer the suspension onto a RTA Spin Filter Set Wait for 1 minute then centrifuge for 2 minutes at 11 000 x g 11 000 rpm Take the Spin Filter out of the Receiver Tube and discard the Receiver Tube 3 First washing of the RTA Spin Filter Place the Spin Filter into a new 2 0 ml Receiver Tube and add 600 ul Wash Buffer SL to the Filter Centrifuge at 11 000 x g 11 000 rpm for 1 minutes Take the Filter from the Receiver Tube discard the filtrate 4 Second washing of the RTA Spin Filter Place the Spin Filter back into the 2 0 ml Receiver Tube and add 700 ul 70 Ethanol to the Filter Centrifuge at 11 000 x g 11 000 rpm for 1 minute Take the Filter from the Receiver
7. procedure Do not eat drink smoke or chew gum at least 30 min prior to saliva collection Collecting 2 ml of saliva may require several minutes Putting some grains of sugar on the tongue helps to produce saliva and does not interfere with the stabilization 1 LY Remove seal from tube completely and discard the seal or unscrew lid from tube and put it aside for further use Z iT I l Insert funnel into tube tightly 3 _ y U Rub cheeks against teeth intensely for 30 sec 4 A Collect saliva to indicated fill level avoid making and f measuring air bubbles lt eus Remove and discard the funnel Press lid firmly on the tube until it clicks or screw lid tightly onto the tube again Gently shake tube for 15 sec to dissolve white reagent o ET x Store the tube upright for 2 20 min with occasional shaking until SalivaGene reagent is dissolved Some cloudy material may occur during this process This does not interfere with stabilization 74 X 11 PSP SalivaGene DNA Kit 0615 PSP Treatment with SalivaGene Collection Module II Intended Use Collection of human saliva samples for DNA stabilization and extraction using the SalivaGene DNA Extraction Kits The DNA in SalivaGene stabilized samples is stable for 1 year at room temperature within the expiry date The product is intended for use under supervision of professionals only such as technicians physician
8. 7 p a ACS ISO 2 Propanol f r die Molekularbiologie 2 Propanol Ordering No 6752 Ordering No A3928 Ordering No 59304 1L F Important indications 1 Process only as many samples as the microcentrifuge allows to process 2 Sample and buffers should be thoroughly mixed and should have room temperature 3 The elution can be done by using lower amount of Elution Buffer This may result in a higher concentration of DNA But pay attention that minimum volume for elution is 30 ul but this will reduce the yield Elution volume between 2 x 50 ul up to 200 ul will realize comparable results 4 The eluted DNA volume can be lower than the added Elution Buffer volume Elution Buffer should be preheated to 48 C 5 Elution Buffer doesn t contain EDTA 6 The yield can be increased if the incubation time with preheated Elution Buffer will be prolonged 10 PSP SalivaGene DNA Kit 0615 PSP Treatment with SalivaGene Collector and Collector Premium Intended Use Collection of human saliva samples for DNA stabilization and extraction For research use only The DNA in SalivaGene stabilized samples is stable for 1 year at room temperature within the expiry date The product is intended for use under supervision of professionals only such as technicians physicians and biologist trained in molecular biological techniques Contents SalivaGene funnel lid tube each tube contains approx 150 mg of SalivaGene reagent Limitations of the
9. Elution Buffer The eluted DNA is ready for use in different downstream applications Yield and quality of DNA The amount of purified DNA in the PSP SalivaGene DNA Kit procedure from different samples depends on the sample type and sample source Yield and quality of isolated DNA is suitable for any molecular diagnostic detection system The diagnostic tests should be performed according to manufacturer s specifications 8 PSP SalivaGene DNA Kit 0615 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 pipette tips between liquid transfers To avoid cross contamination we recommend the use of aerosol barrier pipette tips o When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles Discard contaminated gloves Do not combine components of different kits unless the lot numbers are identical Avoid microbial contamination of the kit reage
10. add 30 ml 30 ml 1035203300 Wash Buffer II add 42 ml 18 ml 1035203400 Elution Buffer 15 ml 1035204000 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 19 PSP SalivaGene DNA Kit 0615 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 2 stratec com www stratec com 1A3g01 06 2015
11. amount of Binding Buffer A insufficient mixing with mix sample with Binding Buffer A by Binding Buffer A pipetting up and down 4 5 times or by vortexing 5 sec prior to transfer the sample onto filter low percentage alcohol used repeat purification procedure with a new sample incomplete elution increase incubation time with preheated Elution Buffer to 5 10 min elute twice with each 100 ul Elution Buffer use higher volume of Elution Buffer low sample DNA concentration elute the DNA with lower volume of Elution Buffer pH of water incorrect acidic low pH may reduce DNA yield Ensure that the pH of the water is at least 7 0 or use Elution Buffer contains only 10 mM Tris HCL no EDTA degraded old material with several freeze avoid repeated thawing and freezing of the material or sheared thaw cycles DNA clogged insufficient lysis perform isolation as described in protocols Spin Filter too much starting material increase lysis time with lysis buffer SalivaGene DNA Stabilize increase centrifugation time and or speed reduce amount of starting material problems ethanol in the eluted DNA verify if the recommended centrifugation time was reached with increase centrifugation time for the elimination of ethanol if subsequent necessary applications e g in PCR salt in the eluate Wash Buffer should be stored and used at RT verify Wash Buffer on the precipitation of salt If there are precipitations dissolve this by careful warming up to 30 C red
12. aring instructions Mix all completely by vortexing 3 Binding of the DNA Transfer the whole suspension onto a RTA Spin Filter Set Incubate for 1 minute at RT Centrifuge for 2 minutes at 11 000 x g 11 000 rpm Take the Spin Filter out of the RTA Receiver Tube and discard the RTA Receiver Tube 4 Washing of the Spin Filter Place the Spin Filter into a new RTA Receiver Tube and add 600 ul Wash Buffer SL to the Filter Centrifuge at 11 000 x g 11 000 rpm for 1 minute Discard filtrate Add 700 ul 70 Ethanol to the Filter Centrifuge at 11 000 x g 11 000 rpm for 1 minute Discard filtrate Repeat the second washing step Discard filtrate Remove ethanol by centrifugation for 4 minutes at maximum speed 5 Elution of DNA Place the RTA Spin Filter into a 1 5 ml Receiver Tube and add 50 100 yl of prewarmed Elution Buffer Incubate for 1 minute at room temperature Close the Spin Filter and centrifuge for 1 minute at 11 000 x g 11 000 rpm 14 PSP SalivaGene DNA Kit 0615 Protocol I Human DNA extraction from stabilized material by the PSP SalivaGene DNA Kit Please read the instructions carefully and conduct the prepared procedure Important Transfer the needed amount of Elution Buffer into 2 0 ml Receiver Tube not included in the kit and place the tube at 48 Attention Please be aware that you have to prepare the Binding Buffer A see instruction page 9 1 Protein removal Transfer 500 ul of
13. aser 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 PSP SalivaGene DNA Kit 4 PSP SalivaGene DNA Kit 0615 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 PSP SalivaGene DNA 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 2903 2907 or contact your local distributor Intended use of the PSP SalivaGene DNA Kit The PSP SalivaGene DNA Kit is suitable for reproducible purification of total DNA from 500 ul stabilization media of swab and saliva samples in forensic human identity genetic bios
14. cally 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 PSP 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 PSP SalivaGene DNA Kit 0615 Contents Kit content of the PSP SalivaGene DNA Kit Kit content of the PSP SalivaGene DNA Kit Content of SalivaGene Collection Sets Symbols Storage Quality control and product warranty Intended use of the PSP SalivaGene DNA Kit Product use limitation Safety information Product characteristic of the PSP SalivaGene DNA Kit Sampling and sample storage Principle and procedure Yield and quality of DNA Important points before starting a protocol Preparing reagents and buffers Reagents and equipment to be supplied by user Important indications PSP Treatment with SalivaGene Collector Premium SalivaGene Collector PSP Treatment with SalivaGene Collection Module II PSP Treatment of SalivaGene Buccal Swab Scheme Protocol Human DNA extraction from stabilized material by the PSP SalivaGene DNA Kit Protocol II Bacterial DNA extraction from stabilized material by the PSP SalivaGene DNA Kit Troubleshootin
15. cap Prepare 500 ml of 7096 Ethanol Incubate the needed amount of Elution Buffer at 48 C 3 PSP SalivaGene DNA Kit 0615 Content of SalivaGene Collection Sets SalivaGene Collection Sets 50 pieces 8 x 50 pieces SalivaGene Buccal Swab 1035230300 SalivaGene Collection Module II 1035212300 SalivaGene Collector Premium SalivaGene Collector Symbols Lot number Catalogue number Expiry date CH Consult operating instructions X Temperature limitation Do not reuse Storage All buffers and kit components except the dissolved Proteinase K of the PSP SalivaGene DNA Kit should be stored well sealed and dry at room temperature and are stable for at least 12 months under these conditions Room temperature RT is defined as range from 15 30 C Dissolved Proteinase K must be stored at 20 C Repeated freezing and thawing will reduce the activity of Proteinase K dramatically Dividing of Proteinase K into aliquots and storage at 20 C is recommended Binding Buffer A charged with Isopropanol Wash Buffer SL charged with Ethanol should be appropriate sealed Before every use make sure that all components have room temperature If there are any precipitates within the provided solutions solve these precipitates by warming carefully Quality control and product warranty STRATEC Molecular warrants the correct function of the PSP SalivaGene DNA Kit for applications as described in this manual Purch
16. e 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 5 Elution of DNA Place the Spin Filter into a new 1 5 ml Receiver Tube and add 50 100 ul of prewarmed Elution Buffer Incubate for 1 min at room temperature Close the Spin Filter 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 30 ul for gram positive bacteria not included in the kit 16 PSP SalivaGene DNA Kit 0615 Troubleshooting Problem Cause Comments and suggestions low amount insufficient cell lysis increase lysis time reduce amount of starting material of DNA overloading of Spin Filter reduces yield insufficient cell lysis due to repeat the DNA purification procedure with a new sample and decreased Proteinase K freshly prepared Proteinase K stock solution Be sure to store activity the stock solution at 20 C insufficient lysis time the DNA purification procedure should be done not earlier than 1 hour after sampling inefficient binding of DNA to overloading RTA Spin Filter reduces yield the membrane e g due to use correct
17. e is completed by a very efficient and fast isolation method up to 500 ul about 30 min which results in high quality total DNA This kit has been designed for isolation of DNA from host organism as well as for DNA from pathogenic bacteria and eukaryotes for in vitro diagnostic analysis After sample collection the saliva or swab sample it is transferred as described in one of the available SalivaGene Collection Sets prefilled with SalivaGene DNA Stabilizer The raw material is lysed under specific conditions The SalivaGene DNA Stabilizer effects inactivation of DNases and prevents degradation of DNA it preserves the microorganism titre and prelyses bacteria The stabilized sample may be shipped in the SalivaGene DNA Stabilizer containing tubes during 12 month at ambient temperature The sample may be stored under this buffer at 20 for a several years For the DNA extraction an aliquot may be used the residual sample is stored for further extractions 500 ul aliquots of the sample are used in the DNA isolation procedure At first the proteins are degraded with Proteinase K followed by a binding of the DNA to a Spin Filter while contaminants pass this filter The DNA is eluted in either water or Elution Buffer provided with the kit Yields are from sample to sample depending on factors as the nature of the sample swab or saliva and the DNA contents of the respective samples The Kit procedure requires neither phenol chloroform e
18. ecurity and pathogen and for in vitro diagnostic analyses The purification is very efficient and the isolated DNA performs well in downstream applications such as quantitative PCR and STR analysis with high signal to noise ratios The system consists four modules three collection sets and one extraction module useable independently or in combination Also included is an easy to handle protocol for DNA purification by precipitation 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 should be interpreted with regard to other Clinical or laboratory findings To minimize irregularities in diagnostic results adequate controls for downstream applications should be used The kit is in compliance with EU Directive 98 79 EC on in vitro medical devices But it is not for in vitro diagnostic use in countries where the EU Directive 98 79 EC on in vitro medical devices is not recognized Product use limitation The kit is neither validated for the isolation of genomic DNA from serum plasma fungi nor from parasites or isolation and purification of total RNA
19. g Appendix General notes on handling DNA Ordering information O co O Oo OO OC OC BR aA FW w 10 11 12 13 14 15 16 17 18 18 19 2 PSP SalivaGene DNA Kit 0615 Kit content of the PSP SalivaGene DNA Kit Store dissolved Proteinase K at 20 C Store all other kit components at room temperature RT Uses so onrctone 0 entacions me a IE Dilute Proteinase K with Add 11 ml 99 7 Add 21 ml 99 796 Initial steps 250 ul ddH2O Mix thoroughly until completely dissolving and store at 20 C Prepare 10 ml of 7096 Ethanol Incubate the needed amount of Elution Buffer at 48 C Isopropanol to the bottle Binding Buffer A Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times Dilute Proteinase K with 1 1 ml ddH20 Mix thoroughly until completely dissolving and store at 20 C Add 36 ml of 96 100 Ethanol to the bottle Wash Buffer SL mix thoroughly and store with tightly closed cap Prepare 100 ml of 70 Ethanol Incubate the needed amount of Elution Buffer at 48 C Isopropanol to each bottle Binding Buffer A Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times Dilute each tube Proteinase K with 1 1 ml ddH20 Mix thoroughly until completely dissolving and store at 20 C Add 120 ml of 96 100 Ethanol to the bottle Wash Buffer SL mix thoroughly and store with tightly closed
20. g and store at 20 C Add 36 ml of 96 100 Ethanol to Wash Buffer SL mix thoroughly and keep the bottle always firmly closed Prepare 100 ml of 70 Ethanol 250 DNA extractions Add 21 ml 99 795 Isopropanol to the Binding Buffer A Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times Dilute each tube Proteinase K with 1 1 ml ddH5O Mix thoroughly until completely dissolving and store at 20 C Add 120 ml of 96 100 Ethanol to Wash Buffer SL mix thoroughly and keep the bottle always firmly closed Prepare 500 ml of 70 Ethanol 9 PSP SalivaGene DNA Kit 0615 Reagents and equipment to be supplied by user When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information please consult the appropriate material safety data sheets MSDS These are available online in convenient and compact PDF format at www stratec com under each STRATEC Molecular kit and kit component Microcentrifuge Thermomixer for 48 C Measuring cylinder 250 ml Disposable gloves Pipette and pipette tips Vortexer Reaction tubes 1 5 ml or 2 0 ml dd H2O 96 100 Ethanol Isopropanol 00000000000 The PSP SalivaGene DNA 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
21. llection Sets into a 1 5 ml reaction tube not provided Add 10 ul of Lysozyme 10 mg ml and incubate for 10 min at 37 C under continuously shaking or every 5 minute flicking the tube Add 20 ul of Proteinase K and incubate for 20 min at 48 C with shaking or every 5 minute flicking of the tube Incubate the sample for additional 10 minutes under continuously shaking at 95 C This step will lead to a thermic disintegration of bacterial cell wall structures Human Genomic DNA of the cellular material might be broken by this step 2 Binding of DNA to the RTA Spin Filter Add 200 ul of Binding Buffer A to the 1 5 ml reaction tube mix well Transfer the suspension onto a RTA Spin Filter Set Wait for 1 minute Then centrifuge for 2 minutes at 11 000 x g 11 000 rpm Take the Spin Filter out of the Receiver Tube and discard the Receiver Tube 3 First washing of the RTA Spin Filter Place the Spin Filter into a new 2 0 ml Receiver Tube and add 600 ul Wash Buffer SL to the Filter Centrifuge at 11 000 x g 11 000 rpm for 1 minute Take the Filter from the Receiver Tube discard the filtrate 4 Second washing of the RTA Spin Filter Place the Spin Filter back into the 2 0 ml Receiver Tube and add 700 ul 70 Ethanol to the Filter Centrifuge 11 000 x g 11 000 rpm for 1 minute 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 Discard the filtrat
22. nts To minimize the risk of infections from potentially infectious material we recommend working under laminar airflow until the samples are lysed o This kit should only be used by trained personnel Ooo0o o0 Preparing reagents and buffers 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 Adjust the Thermomixer to 48 C Warm up the needed amount of Elution Buffer to 48 C up to 100 ul Elution Buffer are needed per sample Label the needed amount of RTA Spin Filter lid Label the needed amount of 1 5 ml Receiver Tubes per sample 1 Receiver Tube Add the needed ul ddHO to reaction tube with Proteinase K see below Vortex for 5 s Add the needed amount of Isopropanol to the Binding Buffer A Vortex for 5 s Add the needed amount of Ethanol to the Wash Buffer SL Vortex for 5 s Prepare the needed amount of 7096 Ethanol No ONDARY 5 DNA extractions Add 250 ul ddH O to Proteinase K mix thoroughly until completely dissolving and store at 20 C Binding Buffer A and Wash Buffer SL are ready to use Prepare 10 ml of 70 Ethanol 50 DNA extractions Add 11 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 Dilute the Proteinase K with 1 1 ml ddH2O Mix thoroughly until completely dissolvin
23. on 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 18 PSP SalivaGene DNA Kit 0615 Ordering information Product Package Size Catalogue No PSP SalivaGene DNA Kit 5 preparations 1035200100 PSP SalivaGene DNA Kit 50 preparations 1035200200 PSP SalivaGene DNA Kit 250 preparations 1035200300 SalivaGene Collector Premium 5 pieces 1035210100 SalivaGene Collector Premium 50 pieces 1035210200 SalivaGene Collection Module II 5 container 1035212100 SalivaGene Collection Module II 50 container 1035212200 SalivaGene Collection Module II 125 container 1035212300 SalivaGene Buccal Swab 5 pieces 1035230100 SalivaGene Buccal Swab 50 pieces 1035230200 PSP SalivaGene Precipitation Kit 50 preparations 1035290200 SalivaGene DNA Stabilizer 30 ml 1035201100 Binding Buffer A add 21 ml 9 ml 1035202100 Wash Buffer
24. ratory 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 P273 Avoid release to the environment 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 PSP SalivaGene DNA Kit 0615 Product characteristic of the PSP SalivaGene DNA Kit Starting material Yield Time Ratio 500 ul of stabilization buffer SalivaGene UP to 10 ug Paso A 280 DNA Stabilizer for all 3 Saliva Collection depends of kind of starting RETAN Sets material 500 ul of swab stabilization SalivaGene up to 1 ug DNA Stabilizer depends on the donor The PSP SalivaGene DNA Kit uses the cost efficient PSP Technology Preanalytical Sample Processing It is a modular designed system for collection stabilization storage and transportation of saliva or swab samples completed with DNA purification The system combines the use of prefilled SalivaGene Collection Sets for saliva or swab sample collection with the storage and stabilization of respective specimen The DNA is protected during transportation Bacterial samples are prelysed bacterial growth is inhibited The procedur
25. s and biologist trained in molecular biological techniques Contents Collection tube Stabilizer tube The stabilizer tube contains approx 2 ml of liquid Saliva DNA Stabilizer reagent Limitations of the procedure Do not eat drink smoke or chew gum at least 30 min prior to saliva collection Collecting 2 ml of saliva may require several minutes Putting some grains of sugar on the tongue helps to produce saliva and does not interfere with the stabilization Unscrew lid from Collection tube and put it aside for further use Rub cheeks against teeth intensely for 30 sec Collect saliva to indicated fill level avoid making and measuring air bubbles Unscrew and discard lid from Stabilizer tube Pour Saliva DNA Stabilizer reagent into Collection tube Screw lid tightly onto the Collection tube again Shake tube for 15 sec to mix saliva and Saliva DNA Stabilizer reagent 12 PSP SalivaGene DNA Kit 0615 PSP Treatment of SalivaGene Buccal Swab Intended Use Collection of human oral samples for DNA stabilization and extraction using the SalivaGene DNA Extraction Kits The DNA in SalivaGene stabilized samples is stable for 1 year at room temperature within the expiry date The product is intended for use under supervision of professionals only such as technicians physicians and biologist trained in molecular biological techniques Contents Swab Stabilizer tube label for donor description The Stabilizer tube contains
26. ts 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 PSP SalivaGene DNA 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 European Community risk and safety phrases for the components of the PSP SalivaGene DNA Kit and for the SalivaGene Stabilization Sets to which they apply are listed below as follows Proteinase K Saliva DNA Stabilizer danger danger H315 319 334 335 P280 305 351 338 310 405 H319 P305 351 338 H319 Causes serious eye irritation 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 H315 Causes skin irritation H334 May cause allergy or asthma symptoms or breathing difficulties if inhaled H335 May cause respi
27. uced sensitivity of amplification adjust the volume of eluate added as template in the reaction amplification reaction A260 A280 rati insufficient cell lysis and protein see above under low amount of DNA for purified degradation due to decreased DNA is low Proteinase K activity insufficient lysis due to insufficient see above under low amount of DNA Proteinase K activity 17 PSP SalivaGene DNA Kit 0615 Appendix General notes on handling DNA Starting material This kit is designed for extraction of DNA from saliva and swabs These materials show big variation in DNA contents The purification of some apoptotic DNA is normal Nature of DNA The length and delicate physical nature of DNA requires careful handling to avoid damage due to shearing and enzymatic degradation Other conditions that affect the integrity and stability of DNA include acidic and alkaline environments high temperature and UV irradiation Careful isolation and handling of high molecular weight DNA is necessary to ensure compatibility with various downstream applications Damaged DNA could perform poorly in applications such as genomic Southern blotting and long template PCR Storage of DNA A working stock of DNA can be stored at 2 4 C for several weeks For long term storage DNA should be stored at 20 C but storing at 20 C can cause shearing particularly if the DNA is exposed to repeated freeze thaw cycles Note that the soluti
28. xtraction nor alcohol precipitation A minimal interaction by the user allows safe handling of potentially infectious samples The whole process is designed to avoid sample to sample cross contamination Due to the high purity the isolated genomic DNA is ready to use for a broad panel of downstream applications see below or can be stored at 20 C for subsequent use o PCR Real Time PCR Analysis RFLP AFLP Analysis o Restriction Enzyme Digestion o HLA Typing o Pathogen Detection For an higher yield of DNA use our easy to handle PSP SalivaGene Precipitation Kit order no 1035290200 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 PSP SalivaGene DNA Kit cannot be construed as an authorization or implicit licence to practice PCR under any patents held by Hoffmann LaRoche Inc 7 PSP SalivaGene DNA Kit 0615 Sampling and sample storage Starting material The amount of starting material used in PSP SalivaGene DNA Kit procedures can vary slightly depending on the amount of DNA present in the sample Specific guidance for starting amounts is given in the individual protocols Swab Saliva The protocol works with aliquots of transport media for collection of fresh saliva or swab samples The DNA is stable for at least 12 month at room temperature in the transport media SalivaGene DNA Stabilizer The aliquots of the stabilization buffer may be processed
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