RTP Mycobacteria Kit User manual - Negev Bio
Contents
1. cerebrospinal fluid synovial fluid or for DNA from stool sample parasites or the purification of total RNA The application of the kit for isolation and purification of viral DNA has not been evaluated 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 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 5 RTP Mycbacteria2. starting material Bacterial cultures Bacterial cultures grow in the presence of a selective agent such as an antibiotic The yield and quality of DNA may depend on factors such host strain inoculation antibiotic and type of culture medium The bacteria will be pelleted after cultivation Best results are obtained with fresh material or material that has been immediately frozen and stored at 20 C or 80 C Repeated freezing and thawing of stored samples should be avoided since this leads to reduced DNA size Tissue Biopsy Material Best results are obtained with fresh material or material that has been immediately frozen and stored at 20 C or 80 C Repeated freezing and thawing of stored samples should be avoided since this leads to reduced DNA size Use of poor quality starting material also leads to reduced length and yield of purified DNA The amount of purified DNA from max 10 mg tissue sample depends on the nature of starting material Sample preparation Pre Extraction Processing For sputum sample a pretreatment with NAC Buffer is necessary The sample will be incubate and centrifuged to get a bacteria pellet This pellet will be resuspend with Resuspension Buffer R Lysis Samples are lysed under anti chaotropic conditions at different elevated temperature and continuously shaking Lysis is performed in the presence of Lysozym to break the cell wall of the bacteria a Lysis Buffer and Proteinase K to digest the pr
3. the Extraction Tube L into a Thermomixer and incubate under continuously shaking for 30 60 min minutes at 52 C Lysis time can be increased if the lysis is not completely 2 Lysis at 95 C for 15 min in a Thermomixer Place the Extraction Tube L into a Thermomixer and switch for 95 C for 15 min under continuously shaking continuous shaking increases the lysis efficiency 3 Incubation on ice for 1 min Place the Extraction Tube L on ice for 1 minute Open the Extraction Tube L carefully and add 20 ul Proteinase K 4 Lysis at 65 C for 10 min in a Thermomixer Place the Extraction Tube L into a Thermomixer and incubate at 68 C for 10 min under continuously shaking continuous shaking increases the lysis efficiency 5 Realisation of optimal binding condition Add 400 ul Binding Solutionto the sample and vortex shortly 6 DNA Binding on RTA Spin filter surface Load the sample onto the RTA Spin Filter Set and incubate for 2 min Centrifuge at 11 000 x g 11 000 rpm in a standard table centrifuge for 1 min Discard the filtrate and place the RTA Spin Filter back into the RTA Receiver Tube 7 Washing Add 500 ul Wash Buffer and centrifuge at 11 000 x g 11 000 rpm for 1 min Discard the filtrate place the RTA Spin Filter again into the RTA Receiver Tube 8 Washing Il Add 800 ul Wash Buffer II and centrifuge at 11 000 x g 11 000 rpm for 1 min Discard the filtrate place the RTA Spin Filter back into the RTA Receiver Tube a
4. 043 354 US 6 037 465 EP 0880535 WO 9728171 WO 9534569 EP 0765335 DE 19506887 DE 10041825 2 WO 0034463 Invisorb and RTP are registered trademarks of STRATEC Biomedical AG The PCR process is covered by US Patents 4 683 195 and 4 683 202 and foreign equivalents owned by Hoffmann La Roche AG 2013 STRATEC Molecular all rights reserved 1 RTP Mycbacteria Kit 0213 Contents Kit contents of RTP Mycobacteria Kit Symbols Storage Quality control and product warranty Intended use Product use limitations Safety information Product characteristic of RTP Mycobacteria Kit Principle and procedure Sampling and storage of starting material Sample preparation Yield and quality of genomic DNA Important notes Important points before starting a protocol Internal control IC Extraction control gt O O O O O MW N Oa a FP BB LP W Preparing reagents and buffers Equipment and reagents to be supplied by user 10 Important indication 10 Scheme of RTP Mycobacteria Kit 11 Instructions Protocol 1 Isolation of Mycobacterial DNA from Sputum Sample 200 ul sample volume 12 Protocol 2 Isolation of Mycobacterial DNA from Bronchoalveolar lavage sample 13 Protocol 3 Isolation of Mycobacterial DNA from Sputum Sample of larger volumes up to 5 ml 14 Protocol 4 Isolation of Mycobacterial DNA from tissue biopsies e g lymph notes 15 Troubleshooting 16 Appendix 17 General notes on handling DNA 17 Ordering
5. Kit KEmL 75 purifications 2433150200 InviMag Bacteria DNA Mini Kit KF96 1 x 96 purifications 7433300100 InviMag Bacteria DNA Mini Kit KF96 5 x 96 purifications 7433300200 Invisorb Universal Bacteria HTS 96 Kit X 2 x 96 purifications 7133310200 Invisorb Universal Bacteria HTS 96 Kit X 4 x 96 purifications 7133310300 Invisorb Universal Bacteria HTS 96 Kit X 24 x 96 purifications 7133310400 Possible suppliers for lsopropanol Fa Carl Roth 2 Propanol Fa Applichem Fa Sigma Rotipuran gt 99 7 p a ACS ISO 2 Propanol fur die Molekularbiologie 2 Propanol Ordering No 6752 Ordering No A3928 Ordering No 59304 1L F Possible suppliers for Centrifuges Eppendorf AG SIGMA Laborzentrifugen GmbH 22331 Hamburg Germany 37507 Osterode am Harz Germany Phone 49 0 40 53801 0 Phone 49 5522 5007 0 Fax 49 0 40 53801 556 Fax 49 5522 5007 12 E Mail eppendorf eppendorf com E Mail info sigma zentrifugen de Internet www eppendorf com Internet www sigma zentrifugen de 18 RTP Mycbacteria Kit 0213 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 1A3m01 02 2013
6. Kit 0213 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 RTP Mycobacteria 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 RTP Mycobacteria Kit to which they apply are listed below as follows Wash Buffer I warning H302 312 332 412 EUH032 P273 Extraction Tube L Proteinase K danger danger H302 314 EUH208 P260 280 305 351 338 310 H315 319 334 335 P280 305 35 1 338 3 10 405 H319 Causes serious eye irritation H302 Harmful if swallowed H312 Harmful in contact with skin H332 Harmful if inhaled H412 Harmful to
7. P Mycobacteria Kit allows rapid and efficient isolation of high quality DNA from Mycobacteria species from sputum or bronchalveolar sample or tissue biopsies using the RTP technology This kit technology yields mycobacterial DNA from different human samples that is free of proteins nucleases and other impurities and is ready to use for different downstream applications such as PCR quantitative PCR and others The purified DNA can be used for in vitro diagnostic analysis only THE PRODUCT IS INDENTED FOR USE BY PROFESSIONAL USERS 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 cell free body fluids like
8. Receiver Tube add 800 ul Wash Buffer II centrifuge at 11 000 x g 11 000 rpm for 1 min discard the filtrate place the RTA Spin Filter back into the RTA Receiver Tube centrifuge for 4 min at max speed to remove the ethanol completely place the RTA Spin Filter into a new 1 5 ml Receiver Tube add 80 100 ul of prewarmed Elution Buffer incubate for 1 min at room temperature centrifuge for 1 min at 11 000 x g 11 000 rpm 11 RTP Mycbacteria Kit 0213 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 Isolation of mycobacterial DNA from Sputum Sample 200 ul sample volume Important Heat heating blocks e g Thermomixer to 65 C prewarm the needed amount of Elution Buffer at 65 8 C Be aware of risk of infection when handling material containing MYCOBACTERIA 1 Pre Extraction Processing Transfer 200 ul of sputum into a 1 5 ml reaction tube and add 200 ul of NAC Buffer Vortex shortly and incubate the sample at room temperature under continuously shaking for 20 minutes Centrifuge the sample at 9 300 x g 10 000 rpm for 15 min Remove the supernatant carefully but completely Resuspend the resulting pellet with 400 ul Resuspension Buffer R Transfer the sample completely into the Extraction Tube L Vortex the tube shortly 2 Lysis at 95 C f
9. aquatic life with long lasting effects EUH032 Contact with acids liberates very toxic gas H314 Causes severe skin burns and eye damage EUH208 Contains Proteinase K May produce an allergic reaction 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 P273 Avoid release to the environment P260 Do not breathe dust fume gas mist vapours spray 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 RTP Mycbacteria Kit 0213 Product characteristic of the RTP Mycobacteria Kit Mycobacterial DNA from max 200 ul depends on the 60 75 min A260 A 280 Sputum Sample starting material incl lysis time 1 7 2 0 Mycobacterial DNA from Bronchalveolar Lavage Sample Mycobacterial DNA from max up to 5 ml Sputum Sample Mycobacterial DNA from tissue biopsies e g lymph notes The RTP Mycobacteria DNA Mini Kit allows rapid and efficient isolation of high quality genomic DNA from Mycobacteria included in different kin
10. ds of starting materials The kit uses the patented RTP technology whose special feature is the utilization of an Extraction Tube L containing pre formulated solid lysis reagent lytic enzyme Proteinase K carrier nucleic acid and a precisely calibrated amount of an internal DNA Extraction Control The internal control is a convenient tool for the assessment and monitoring of extraction efficiency and PCR amplification as well for the quality of the purified nucleic acid and for the exclusion of false negative results Using the RTP Mycobacteria Kit all types of samples are transferred into the Extraction Tubes together with a specially designed Resuspension Buffer R The prefilled buffer and enzymes lyse the samples stabilize nucleic acid and enhance the selective DNA adsorption to the membrane The membranes are efficiently washed before the nucleic acid is eluted In addition to the rigorous lysis procedure simple pre treating steps have been introduced ideally for purification of genomic DNA of different sources High extraction efficiency and detection sensitivities will be realized No phenol chloroform extraction is required The hands on time necessary for the whole procedure is reduced to a minimum All kit components can be stored at room temperature The procedures require minimal interaction by the user allowing safe handling of potentially infectious samples Due to the high purity the isolated total DNA is rea
11. dy to use for a broad panel of downstream applications see below or can be stored at 80 C for subsequent use PCR Real time PCR quantitative RT PCR like TaqMan und LightCycler technology RE Digestion RFLP Analysis Sequencing O O O 0 0 To purify high chromosomal bacterial DNA in 96 format STRATEC Molecular offers the Invisorb Universal Bacterial HTS 96 Kit for use in a centrifuge on a vacuum manifold and on common laboratory automated workstations Furthermore STRATEC Molecular offers the InviMag Bacteria DNA Mini Kit KFmL and KF96 for DNA isolation using magnetic beads For further information please contact Tel 49 0 30 9489 2901 2910 in Germany and from foreign countries Tel 49 0 30 9489 2907 or your local distributor 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 RTP Bacteria DNA Mini Kit cannot be construed as an authorization or implicit license to practice PCR under any patents held by Hoffmann LaRoche Inc 7 RTP Mycbacteria Kit 0213 Principle and procedure The RTP Mycobacteria Kit procedure comprises following steps Pre Extraction Processing lysis at different temperatures binding the nucleic acids in the lysate to the membrane of a RTA Spin Filter washing of the membrane and elimination of contaminants and ethanol elution of nucleic acid O OTO O 0 This manual contains 4 protocols Sampling and storage of
12. eam analysis with NAT techniques for examples qPCR RT qPCR LAMP LCR Diagnostic assays should be performed according to the manufacturer s instructions Quantitative RT PCR is recommended for determination of mycobacterial DNA yield 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 at room temperature o When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles o Discard gloves if they become contaminated o Do not combine components of different kits unless the lot numbers are identical o Avoid microbial contamination of the kit reagents o To minimize the risk of infections from potentially infectious material we recommend working under laminar air fl
13. g fluorescent dyes may lead too low results This is caused by the nature of the isolated DNA due the denaturing step 95 C during lysis DNA is partially single stranded So fluorophores using double stranded DNA will not work correctly 10 RTP Mycbacteria Kit 0213 Scheme of the RTP Mycobacteria Kit a j fn f l arh amp ah 4 Please read the protocols carefully prior to the start of the preparation procedure transfer 200 ul of sputum into a 1 5 ml reaction tube add 200 ul NAC Buffer incubate for 20 min at room temperature under continuously shaking centrifuge at 9 300 x g 10 000 rpm for 15 min remove the supernatant resuspend the pellet with 400 ul Resuspension Buffer R transfer the sample to the Extraction Tube Place the Extraction Tube into a Thermomixer And switch for 95 C for 15 min under continuously shaking place the Extraction Tube L on ice for 1 minute add 20 ul Proteinase K to the Extraction Tube L place the Extraction Tube L into a Thermomixer incubate at 65 C for 10 min under continuously shaking add 400 ul Binding Solution load the sample onto the RTA Spin Filter Set incubate for 2 min centrifuge at 11 000 x g 11 000 rpm for 1 min discard the filtrate place the RTA Spin Filter back into the RTA Receiver Tube add 500 ul Wash Buffer I centrifuge at 11 000 x g 11 000 rpm for 1 min discard the filtrate place the RTA Spin Filter again into the RTA
14. information 18 2 RTP Mycbacteria Kit 0213 Kit contents of RTP Mycobacteria Kit Store all kit components at room temperature RT Store dissolved Proteinase K at 20 C 5 extractions 50 extractions 250 extractions Catalog No 1033220100 1033220200 1033220300 Extraction Tubes L 5 50 5 x 50 NAC Buffer 2 ml 15 ml 60 ml Resuspension Buffer R 2x2ml 30 ml 120 ml Binding Solution 3x1ml empty bottle empty bottle fill with 99 7 lsopropanol ready to use final volume 30 ml final volume 120 ml Proteinase K for 250 ul working for 1 ml for5x1 ml solution working solution working solution 15 ml 30 ml 80 ml West Butter ready to use final volume 60 ml final volume 160 ml 15 ml 18 ml 2x 45 ml Wash Buffer ready to use final volume 60 ml final volume 2 x 150 ml Elution Buffer 2ml 3x2ml 30 ml RTA Spin Filter Set 5 50 5 x 50 1 5 ml Receiver Tubes 5 50 5 x 50 Manual 1 1 1 Initial steps Dilute the Proteinase Dilute the Proteinase K by Dilute the Proteinase K by K by addition of 250 ul ddH20 mix thoroughly and store like described below addition of 1 ml ddH20 mix thoroughly and store like described below Fill 30 ml 99 7 Isopropanol molecular biologic grade into the empty bottle Add 30 ml of 96 100 ethanol to the bottle Wash Buffer I mix thoroughly and keep the bottle always firmly closed Add 42 ml of 96 100 ethanol to
15. ix thoroughly and store like described below 250 bacterial DRNA extractions Fill 120 m 99 7 Isopropanol molecular biologic grade into the empty bottle Add 80 ml of 96 100 ethanol to the bottle Wash Buffer I mix thoroughly and keep the bottle always firmly closed Add 105 ml of 96 100 ethanol to the bottle Wash Buffer II mix thoroughly and keep the bottle always firmly closed Dilute the Proteinase K by addition of 1 ml ddH20 mix thoroughly and store like described below Equipment and reagents to be supplied by user When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information please consult the appropriate material safety data sheets MSDS See our webpage www stratec com Microcentrifuge Eppendorf Thermomixer 68 80 C Measuring cylinder 250 ml Disposable gloves Pipet with tips Reagents reservoirs for multichannel pipets 96 100 ethanol ddH O Isopropanol O O Aa O O Aa 10 0 Q The RTP Mycobacteria Kit is validated with 2 Propanol Rotipuran gt 99 7 p a ACS ISO Order no 6752 from Carl Roth Possible suppliers for Isopropanol Fa Carl Roth 2 Propanol Fa Applichem Fa Sigma Rotipuran gt 99 7 p a ACS ISO 2 Propanol fur die Molekularbiologie 2 Propanol Ordering No 6752 Ordering No A3928 Ordering No 59304 1L F Important indication Measurement of the DNA directly after isolation by intercalatin
16. lification Results incorrect storage of starting material old material ethanol carryover during elution salt carryover during elution ensure that the starting material is fresh or stored under appropriate conditions for long time storage at 20 C avoid repeating thawing and freezing of the material old material often contains degraded DNA increase time for removing of ethanol ensure that Wash Buffer is at room temperature check up Wash Buffer for salt precipitates If there are any precipitates solve these precipitates by warming carefully RNA contaminations of extracted DNA start RNAse A digestion not included in the kit 16 RTP Mycbacteria Kit 0213 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 high molecular weight DNA is necessary to ensure it will function well in various downstream applications Damaged DNA could perform poorly in applications such as genomic Southern blotting long template PCR and construction of cosmid libraries Handling fresh and stored material before the extraction of DNA For the isolation of genomic DNA from cells or tissues use ei
17. nd finally centrifuge for 4 min at max speed to remove the ethanol completely 9 Elution of the DNA Place the RTA Spin Filter into a new 1 5 ml Receiver Tube and add 80 100 ul of prewarmed Elution Buffer Incubate for 1 min at room temperature Centrifuge for 1 min at 11 000 x g 11 000 rpm Optional If a white pellet is visible in the eluate spin the sample again at maximum speed and transfer the supernatant into a clean tube 15 RTP Mycbacteria Kit 0213 Troubleshooting Problem Probable cause Comments and suggestions clogged Spin Filter insufficient lysis and or too much starting material increase lysis time increase centrifugation speed reduce amount of starting material low amount of extracted DNA insufficient lysis incomplete elution insufficient mixing of the sample with Isopropanol increase lysis time at 95 C don t forget the addition of Proteinase K 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 Solutioncompletely by pipetting or by vortexing prior to transfer the sample onto the RTA Spin Filter low concentration of extracted DNA too much Elution Buffer incorrect storage of starting material elute the DNA twice with lower volume of Elution Buffer ensure that the storage of starting material was correctly avoid thawing of the material no Amp
18. ntrifuge at 11 000 x g 11 000 rpm in a standard table centrifuge for 1 min Discard the filtrate and place the RTA Spin Filter back into the RTA Receiver Tube 7 Washing Add 500 ul Wash Buffer and centrifuge at 11 000 x g 11 000 rpm for 1 min Discard the filtrate place the RTA Spin Filter again into the RTA Receiver Tube 8 Washing Il Add 800 ul Wash Buffer II and centrifuge at 11 000 x g 11 000 rpm for 1 min Discard the filtrate place the RTA Spin Filter back into the RTA Receiver Tube and finally centrifuge for 4 min at max speed to remove the ethanol completely 9 Elution of the DNA Place the RTA Spin Filter into a new 1 5 ml Receiver Tube and add 80 100 ul of prewarmed Elution Buffer Incubate for 1 min at room temperature Centrifuge for 1 min at 11 000 x g 11 000 rpm Optional If a white pellet is visible in the eluate spin the sample again at maximum speed and transfer the supernatant into a clean tube 14 RTP Mycbacteria Kit 0213 Protocol 4 Isolation of Mycobacterial DNA from tissue biopsies e g lymph notes Important Heat heating blocks e g Thermomixer to 52 C 68 C and 95 C prewarm the needed amount of Elution Buffer at 68 C Be aware of risk of infection when handling material containing MYCOBACTERIA 1 Pre Extraction Processing Transfer 1mg up to max 10 mg of the biopsy into the Extraction Tube L Add 400 ul of Resuspension Buffer R Close the cap and vortex shortly Place
19. nuously shaking continuous shaking increases the lysis efficiency 5 Realization of optimal binding condition Add 400 ul Binding Solutionto the sample and vortex shortly 6 DNA Binding on RTA Spin filter surface Load the sample onto the RTA Spin Filter Set and incubate for 2 min Centrifuge at 11 000 x g 11 000 rpm in a standard table centrifuge for 1 min Discard the filtrate and place the RTA Spin Filter back into the RTA Receiver Tube 7 Washing Add 500 ul Wash Buffer and centrifuge at 11 000 x g 11 000 rpm for 1 min Discard the filtrate place the RTA Spin Filter again into the RTA Receiver Tube 8 Washing Il Add 800 ul Wash Buffer II and centrifuge at 11 000 x g 11 000 rpm for 1 min Discard the filtrate place the RTA Spin Filter back into the RTA Receiver Tube and finally centrifuge for 4 min at max speed to remove the ethanol completely 9 Elution of the DNA Place the RTA Spin Filter into a new 1 5 ml Receiver Tube and add 80 100 ul of prewarmed Elution Buffer Incubate for 1 min at room temperature Centrifuge for 1 min at 11 000 x g 11 000 rpm Optional If a white pellet is visible in the eluate spin the sample again at maximum speed and transfer the supernatant into a clean tube 13 RTP Mycbacteria Kit 0213 Protocol 3 Isolation of mycobacterial DNA from Sputum Sample of larger volumes up to 5 ml Important Heat heating blocks e g Thermomixer to 65 C prewarm the needed amount of Elu
20. on Buffer Incubate for 1 min at room temperature Centrifuge for 1 min at 11 000 x g 11 000 rpm Optional If a white pellet is visible in the eluate spin the sample again at maximum speed and transfer the supernatant into a clean tube 12 RTP Mycbacteria Kit 0213 Protocol 2 Isolation of Mycobacterial DNA from Bronchoalveolar Lavage Sample Important Heat heating blocks e g Thermomixer to 65 C C prewarm the needed amount of Elution Buffer at 65 C Be aware of risk of infection when handling material containing MYCOBACTERIA 1 Pre Extraction Processing Transfer up to 1 ml of bronchalveolar lavage sample and mix it with up to 1 ml of NAC buffer ratio 1 1 and incubate for 20 min at RT Centrifuge the sample at 9 300 x g 10 000 rpm for 15 min Remove the supernatant carefully but completely Resuspend the resulting pellet with 400 ul Resuspension Buffer R Transfer the sample completely into the Extraction Tube L Vortex the tube shortly 2 Lysis at 95 C for 15 min in a Thermomixer Place the Extraction Tube L into a Thermomixer and switch for 95 C for 15 min under continuously shaking continuous shaking increases the lysis efficiency 3 Incubation on ice for 1 min Place the Extraction Tube L on ice for 1 minute Open the Extraction Tube L carefully and add 20 ul Proteinase K 4 Lysis at 65 C for 10 min in a Thermomixer Place the Extraction Tube L into a Thermomixer and incubate at 68 C for 10 min under conti
21. or 15 min in a Thermomixer Place the Extraction Tube L into a Thermomixer and switch 95 C for 15 min under continuously shaking continuous shaking increases the lysis efficiency 3 Incubation on ice for 1 min Place the Extraction Tube L on ice for 1 minute Open the Extraction Tube L carefully and add 20 ul Proteinase K 4 Lysis at 65 C for 10 min in a Thermomixer Place the Extraction Tube L into a Thermomixer and incubate at 68 C for 10 min under continuously shaking continuous shaking increases the lysis efficiency 5 Realization of optimal binding condition Add 400 ul Binding Solutionto the sample and vortex shortly 6 DNA Binding on RTA Spin filter surface Load the sample onto the RTA Spin Filter Set and incubate for 2 min Centrifuge at 11 000 x g 11 000 rpm in a standard table centrifuge for 1 min Discard the filtrate and place the RTA Spin Filter back into the RTA Receiver Tube 7 Washing Add 500 ul Wash Buffer and centrifuge at 11 000 x g 11 000 rpm for 1 min Discard the filtrate place the RTA Spin Filter again into the RTA Receiver Tube 8 Washing Il Add 800 ul Wash Buffer II and centrifuge at 11 000 x g 11 000 rpm for 1 min Discard the filtrate place the RTA Spin Filter back into the RTA Receiver Tube and finally centrifuge for 4 min at max speed to remove the ethanol completely 9 Elution of the DNA Place the RTA Spin Filter into a new 1 5 ml Receiver Tube and add 80 100 ul of prewarmed Eluti
22. oteins All is provided prefilled in the Extraction Tube L Unlysed sample parts should be removed before the binding step Binding genomic DNA By adding Binding Solutionto the lysate optimal binding condition will be adjusted Each lysate is then applied to a RTA Spin filter and genomic DNA is adsorbed onto membrane as the lysate is drawn through by centrifugal force Removing residual contaminants Contaminants are efficiently washed away using Wash Buffer s while the bacterial genomic DNA remains bound to the membrane Elution of pure genomic DNA Genomic DNA is eluted from the spin filter using 80 100 ul prewarmed Elution Buffer or water Eluting twice each with 80 ul leads to little increase of DNA yield Usage small elution volumes may raise DNA concentration Elution volumes should be at least 50 ul The eluted DNA is ready for use in different downstream applications 8 RTP Mycbacteria Kit 0213 Yield and quality of genomic DNA The amount of purified DNA using the RTP Mycobacteria Kit procedure depends on the sample type and the number of mycobacteria cells in the sample depending from the source e g patients age and health situation transport conditions storage and age of the sample For most samples a single elution with 100 ul Elution Buffer is sufficient Yields of bacterial DNA isolated from biological samples can be small and therefore difficult to determine photometrical The kit is suitable for downstr
23. ow until the samples are lysed o This kit should only be used by trained personnel Internal control IC Extraction control Internal Controls IC from the PCR assay provider can be used as extraction controls if the fragments are longer than 100 bp In this case they have to be added after finalization of the lysis step Attention don t add directly these Internal Controls to the sample 9 RTP Mycbacteria Kit 0213 Preparing reagents and buffers 1 Adjust the thermomixer to 37 C 2 Warm up the needed amount of Elution Buffer to 68 C 80 100 ul Elution Buffer are needed per sample 3 Heat heating blocks e g thermomixer to 68 C and 95 C 4 Label the needed amount of 2 0 ml RTA Spin Filter Sets 5 Label the needed amount of 1 5 ml Receiver Tubes add the needed amount of ethanol to the Wash Buffer I and II see Kit Contents page 3 5 bacterial DNA extractions Binding Solution Wash Buffer I and II are ready to use Dilute the Proteinase K by addition of 250 ul ddH20 mix thoroughly and store like described below 50 bacterial DNA extractions Fill 30 ml 99 7 Isopropanol molecular biologic grade into the empty bottle Add 30 ml of 96 100 ethanol to the bottle Wash Buffer I mix thoroughly and keep the bottle always firmly closed Add 42 ml of 96 100 ethanol to the bottle Wash Buffer II mix thoroughly and keep the bottle always firmly closed Dilute the Proteinase K by addition of 1 ml ddH20 m
24. stratecee molecular User manual RTP Mycobacteria Kit for purification of total DNA from mycobacteria from sputum bronchial lavage and tissue biopsies 101033220X eal STRATEC Molecular GmbH D 13125 Berlin Er Instruction for the RTP Mycobacteria Kit The RTP Mycobacteria Kit allows rapid and efficient isolation of high quality DNA from Mycobacteria species from sputum or bronchalveolar sample or tissue biopsies The RTP Mycobacteria Kit combines an efficient pre analytical sample processing with the efficient isolation of bacterial DNA by binding of bacterial DNA onto a Spin Filter surface by a patented technology without chaotropic components The kit is neither validated for the isolation of genomic DNA from cell free body fluids like cerebrospinal fluid synovial fluid or for DNA from stool sample parasites or the purification of total RNA The application of the kit for isolation and purification of viral DNA has not been evaluated 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 RTP Eppendorf Registered marks trademarks etc used in this document even when not specifically marked as such are not to be considered unprotected by law The Invisorb technology is covered by patents and patent applications US 6 110363 US 6
25. the bottle Wash Buffer Il mix thoroughly and keep the bottle always firmly closed addition of 1 ml ddH20 mix thoroughly and store like described below Fill 120 ml 99 7 Isopropanol molecular biologic grade into the empty Add 80 ml of 96 100 ethanol to the bottle Wash Buffer I mix thoroughly and keep the bottle always firmly closed Add 105 ml of 96 100 ethanol to the bottle Wash Buffer II mix thoroughly and keep the bottle always firmly closed RTP Mycbacteria Kit 0213 Symbols lot number catalog number od date of manufacture expiry date Ci consult operating instructions temperature limitation Q do not reuse Storage The RTP Mycobacteria Kit including the Extraction Tubes L incl Lysis Buffer Proteinase K Carrier RNA Lysozyme and Internal Control DNA should be stored dry at room temperature RT and is stable for at least 12 months under these conditions Wash Buffer I and Wash Buffer II charged with ethanol should be appropriately sealed and stored at room temperature Dividing the Proteinase K into aliquots and storage at 20 C is recommended Storage at 20 C will prolong the activity but repeating freezing and thawing will reduce the activity dramatically Before every use make sure that all components have room temperature If there are any precipitates within the provided solutions dissolve these precipitates by carefully warming up to room temperature Room tempera
26. ther fresh samples or samples that have been quickly frozen in liquid nitrogen and stored at 70 C This procedure minimizes degradation of crude DNA by limiting the activity of endogenous nucleases Storage of DNA Store genomic DNA at 2 to 8 C Storing genomic DNA at 15 to 25 C can cause shearing of DNA particularly if the DNA is exposed to repeated freeze thaw cycles Plasmid DNA and other small circular DNA s can be stored at 2 to 8 C or at 15 to 25 C Handling of DNA 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 17 RTP Mycbacteria Kit 0213 Ordering information Product Package size Catalogue No RTP Mycobacteria Kit 5 extractions 1033220100 RTP Mycobacteria Kit 50 extractions 1033220200 RTP Mycobacteria Kit 250 extractions 1033220300 Single components Extraction Tube L 10 tubes 1033221500 NAC Buffer 15 ml 1033221100 Resuspension Buffer R 30 ml 1033222200 Wash Buffer 30ml 1033223500 Wash Buffer II 18 ml 1033223600 Elution Buffer 15 ml 1033224000 Related Products RTP Bacteria DNA Mini Kit 5 extractions 1033200100 RTP Bacteria DNA Mini Kit 50 extractions 1033200200 RTP Bacteria DNA Mini Kit 250 extractions 1033200300 InviMag Bacteria DNA Mini Kit KEmL 15 purifications 2433150100 InviMag Bacteria DNA Mini
27. tion Buffer at 65 C Note Order additional NAC Buffer under catalog no NAC Buffer 10332211 Be aware of risk of infection when handling material containing MYCOBACTERIA 1 Pre Extraction Processing Transfer up to 5 ml of sputum sample into a 15 ml reaction tube Inactivate the sample by incubation at 65 C for 30 minutes Add equal volume of NAC Buffer vortex shortly and incubate the sample at room temperature for 20 minutes Centrifuge at 960 x g 3 000 rpm for 15 minutes Remove the supernatant carefully but completely Resuspend the resulting pellet with 400 ul Resuspension Buffer R Transfer the sample completely into the Extraction Tube L Vortex the tube shortly 2 Lysis at 95 C for 15 min in a Thermomixer Place the Extraction Tube L into a Thermomixer and switch for 95 C for 15 min under continuously shaking continuous shaking increases the lysis efficiency 3 Incubation on ice for 1 min Place the Extraction Tube L on ice for 1 minute Open the Extraction Tube L carefully and add 20 ul Proteinase K 4 Lysis at 65 C for 10 min in a Thermomixer Place the Extraction Tube L into a Thermomixer and incubate at 68 C for 10 min under continuously shaking continuous shaking increases the lysis efficiency 5 Realization of optimal binding condition Add 400 ul Binding Solutionto the sample and vortex shortly 6 DNA Binding on RTA Spin filter surface Load the sample onto the RTA Spin Filter Set and incubate for 2 min Ce
28. ture RT is defined as range from 15 30 C Quality control and product warranty STRATEC Molecular warrants the correct function of the RTP Mycobacteria 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 RTP Mycobacteria Kit 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 RTP Mycobacteria 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 4 RTP Mycbacteria Kit 0213 Intended use The RT
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