AT Test Pseudomonas aeruginosa
Contents
1. The kit is manufactured for research use only The kit is intended for use by personnel that are well trained in molecular biology Preparation of DNA from P aeruginosa colonies clones requires expertise in microbiology and the local regulations for handling of potentially infectious organisms are to be obeyed Specifications Upon receipt the assay components need to be stored at different temperatures as specified on the package insert The assay is to be performed at an ambient temperature of 18 C to 28 C unless otherwise specified Technical Support CLONDIAG provides technical assistance Please contact Tel 49 0 3641 3111 116 e mail dorit lichter clondiag com Safety Precautions DNA preparation from P aeruginosa must be performed under the biosafety conditions as defined by your local authorities for work with P aeruginosa In most countries biosafety Level 2 is obligatory for this kind of work P aeruginosa DNA may be processed in laboratories below this biosafety level if potential contamination by living P aeruginosa bacteria can be definitively eliminated Always wear protective clothes as regulated for laboratory work by your local authorities Material Safety Data Sheets MSDS Per OSHA 29CFR1910 1200 Commonwealth of Australia NOHSC 1005 1008 1999 and the latest amendments to the European Union Directives 67 548 EC and 1999 45 EC the enclosed reagents do not require a Material Safety Data Sheet2. APPENDIX 3 Re order numbers of individual components component name amount order no o BP J Labelling Buffer 350 pl j 245103001 __ OOS B2 Labelling Enzyme _ 15 pe 245104000 SOSS ci Hybridisation Buffer 60 mL j 245105000 OS c2 ooo Washing Buffer 120 mL 245106000 ee ce HRP Conjugate 100x 150 pL 245107000 ee c4 ooo Conjugate Buffer 30 mt 245108000 a cS o o ooo Washing Buffer2 120 mL J 245109000 SOSEO D1 HRP Substrate 12 mL j 245110000 ArrayTube P a Array P aeruginosa 50 St 201007162 Order number of the P aeruginosa kit 50 reactions 205200050 www clondiag com 05 16 04 0004 V01 24
3. MSDS They do not contain more than 1 of a component classified as hazardous and do not contain more than 0 1 of a component www clondiag com 05 16 04 0004 VO1 4 CLONDIAG classified as carcinogenic MSDS therefore are not provided Nevertheless the buffers may cause irritation if they come into contact with eyes or skin and may cause harm if swallowed The regular precautions associated with laboratory work should be obeyed e g wear protective goggles gloves and lab coat and avoid contact with the reagents In case buffers or solutions are spilled clean with laboratory detergent and water Clondiag assumes no liability for damage resulting from handling or contact with these products If you have any questions please contact our Technical Support see above Shipping Precautions RID ADR Kein Gefahrgut No dangerous goods IMDG No dangerous goods IATA No dangerous goods www clondiag com 05 16 04 0004 V01 5 CLONDIAG KIT COMPONENTS STORAGE AND STABILITY DNA labelling and amplification e B1 Labelling Buffer Store at 2 8 C Surplus 40 e B2 Labelling Enzyme Store at 2 8 C Surplus 200 Hybridisation and Detection e ArrayTubes 50 samples protected against light and sealed under inert gas Store at 18 C to 28 C After opening to be used within two weeks Protect them against humidity and dust and store them at a dark place Avoid ANY touching or scratching the microarray on the bottom of t
4. poor DNA quality or both Staining Control Biotin spots In case that the Biotin spots as described in chapter 8 are negative proceed as follows e Horseradish Peroxidase Conjugate may have degraded during storage Add 1uL buffer C3 4 to 9 uL D1 substrate If the solution turns green within 3 5 seconds the Horseradish Peroxidase still has sufficient enzymatic activity www clondiag com 05 16 04 0004 VO1 19 CLONDIAG e Enzymatic reaction is inhibited by carryover of buffer C1 Ensure proper washing of the tubes to remove all of Buffer C1 prior to adding Horseradish Peroxidase Conjugate Physical damage of the array Scratching of the array surface with a pipette tip can lead to the damage of array spots We recommend to review Section 3 for General Precautions in array handling Weak marker signals but strong Biotin spots Something went wrong with the preparation and or with the labelling of DNA Please check the quality of your DNA preparation with via gel electrophoresis ADDITIONAL INFORMATION Warranty CLONDIAG guarantees the performance as described in this manual Usage of the Kit was successfully tested at ambient temperatures up to 37 C a guarantee is limited to ambient temperatures in the laboratory between 18 C and 28 C Kit components comprise the arrays the reagents for DNA labelling and for detection of labelled DNA products on the array the ArrayMate reader and its software In case one of these comp
5. v discard DNA wash 3x 0 5 ml Buffer C2 2x 30 C 550 rpm 5 min v incubate with conjugate 0 1 ml Buffer C3 4 30 C 550 rpm 10 min lt q discard conjugate rinse 2x 0 5 ml Buffer C5 lt 1 incubate with substrate 0 1 ml Buffer D1 RT 10 min lt q www clondiag com CLONDIAG prepare DNA grow bacteria not part of the kit isolate DNA not part of the kit label DNA 5 ul DNA 4 9 uL B1 0 1 uL B2 Thermocycler add 90 ul of Buffer C1 take image analyse pro cessing time over night 3 4h 3h 2min 2min 60 min 15 min 10 min 2min 10 min 15 min total time requirement over night 05_16 04 0004 VO1 7 8h hands on time 5 min 10 min 5 min 2min 2min 0 min 15 min 2min 2min 10 min 15 min 70 min CLONDIAG 1 P aeruginosa DNA The required specimen for the P aeruginosa genotyping test is 0 5 2 ug of intact DNA from a single colony of P aeruginosa The DNA specimen needs to be virtually free of RNA as determined by agarose gel electrophoresis Please note This assay requires much more DNA than common PCR applications Most performance problems with the P aeruginosa Genotyping Kit are due to insufficient amounts of DNA We therefore strongly recommend to follow the protocol outlined below From several DNA isolation kits tested so far only the DNA extraction Kits from Qiagen in combination with t
6. Blood amp Tissue Kit from Qiagen cat 69504 Instrumentation provided by Clondiag to be ordered separately ArrayMate Reader or ATRO3 reader respectively Materials and instruments required but not provided by Clondiag Equipment growth media and consumables needed for the cultivation of P aeruginosa Equipment needed for DNA isolation e g pipettes centrifuge Kit for isolation of DNA we recommend the Qiagen DNeasy Blood amp Tissue kit Qiagen order number 69506 RNAse we recommend Qiagen s RNase A solution 100 mg mL Qiagen order number 19101 Thermomixer we recommend BioShakelQ from Quantifoil Instruments www ginstruments com equipped with a platform for the warming of 1 5 mL centrifuge tubes Photometer for quality control of DNA Equipment for DNA gel electrophoresis for quality control of DNA Thermocyler Pipettes suitable for 1uL 5uL volumes 90uL 100 uL 200uL 1000uL For removal of liquid from the ArrayTubes we recommend the use of plastic Pasteur pipettes with flexible tips Pasteur pipette plastic with a flexible tip Cee T flexible tip Reagent tubes suitable for PCR ultrapure water www clondiag com 05 16 04 0004 V01 7 PROTOCOL prepare Arrays 4 e rinse arrays 0 5 ml water 60 C 550 rpm 2min e incubate in bufer C1 0 2 ml 60 C 550 rpm 2min e discard C1 process promptly transfer DNA to ArrayTube v hybridise 60 C 550 rpm 60 min
7. Clinical Diagnostics TEST PRINCIPLE Clonal P aeruginosa DNA is amplified approximately 50 fold and labelled with biotin dUTP adopting a so called linear PCR protocol In linear PCR only one primer is used instead of a primer pair producing single stranded reaction products only therefore limiting the degree of amplification limited risk of cross contamination Labelled single stranded DNA is transferred and hybridised to microarrays with 77 probes in duplicate each representing different genetic markers Strain assignment is performed by visual array analysis using the hexadecimal code described by Wiehlmann et al 2007 PNAS vol 104 no 19 p 8101 8106 A next generation kit with an automated software analysis tool is in progress and will be launched by the end of 2010 It will be based on the same chemistry on the same markers and on the same strain assignment strategy using the hexadecimalcode respectively Test results will be 100 comparable to the current visual method THE ASSAY IS DESIGNED to be performed by personnel trained in microbiological and molecular biological laboratory methods Literature e Wiehlmann et al 2004 JOURNAL OF BACTERIOLOGY Vol 186 No 13 p 4228 4237 e Wiehlmann et al 2007 PNAS vol 104 no 19 p 8101 8106 www clondiag com 05 16 04 0004 V01 3 CLONDIAG GENERAL INSTRUCTIONS FOR USE Intended Use For Investigational Use Only Not Intended for Use in Clinical Diagnostics
8. SNP 2 eee eee marker oaeoe r opot eeeerees fliCb ExoS ExoU may be positive 1 or negative 0 respectively like fliCa fliCa SNP www clondiag com 05 16 04 0004 V01 17 CLONDIAG 2 level of interpretation the hexadecimal code As a second step convert the 0 1 string code into the Hexadecimal code as outlined below 0 1 string from 1st level analysis noxe Soxe q l dNS 9 l e l Z dwe nox 9 dwe Sox G dwe q t dwe dNS 9 l e p Z dwe 9 Odwe 9dwe g Odwe L odwe r odwe do dwe Z S 9 L dwe jido c SH9 L SHO L SH9 cayle zgje judo Judo separate it into four subsequent parts and find the four corresponding code digits OHO Quo lt _ pog x a Ho umt nonoo qaou nox Sox q o BurdA 104 yUa oyWNs S EDI lt eSIOA 891A PUL 0 q9 I UOY LEEDI H dNS 9 l e pll a gt QPOD XOHIO K NMYTNORADAQNOOWL Z odwe 9 dwe G dwe y odwe ee ol epog XeH o m mon wo lt DOOM 9dwe L odwe do c SH9 BPOD X9HIO e NMOTwHIORADADAMNOAWL L SH9 caxle judo OHO a E The Hexadecimal code for the SNP pattern of the strain P aeruginosa PAK therefore is SSAA This code may be used for comparison of different strains and it is portable between different laboratories 18 05_16 04 0004 VO1 www clondiag com CLONDIAG TROUBLESHOOTING Data Quality In case of poor data q
9. negative ficaSNP 1 ae fliCa SNP 2 estt oven marker fliCb ExoS ExoU may be positive 1 or negative 0 respectively like fliCa www clondiag com 05 16 04 0004 V01 21 CLONDIAG APPENDIX 2 substances markers marker groups and their respective positions on the array a probes markers for strain assignment probe ma Bai group probe numbers no name name and their positions on the array T a 1 71 71 72 72 67 67 68 68 g PAO Ao non PAO oprL 2 PAO oprL 2 non PAO fliCa SNP 1 PAO fliCa SNP 1 non PAO fliCa SNP 2 PAO fliCa SNP 2 non PAO opri 1 PAO opri 1 non PAO Aa PAO ule non PAO fica exoS U continued see next page www clondiag com 05 16 04 0004 V01 22 CLONDIAG b additional probes and markers continued from previous page probe marker marker group probe numbers no name no no name and their positions on the array 1 71 71 727 variable 67 67 68 6 genes orld Fla Glyk fla island isid orfA 47D7 Isld 2 47D7 IsId 1 PAGI1 XF1753 acetyl transf PA0980 fpvA type fpvA type lla fpvA type II b fpvA type III pKLC unknown pKLC adhesin pKLC f a synthase PKL pkLC102 PAPI pili chaperone group PAPI 1 luminal bd prot 7 PAGI 2 3 1 PAGI 2 3 4 PAGI 2 3 5 PAGI 2 3 6 C 45 5 C 46 52 C 47 53 51 52 PAGI 3 1 55 56 0 PAGI3 8 55 PAGI 2 56 www clondiag com 05 16 04 0004 V01 23 CLONDIAG
10. there MUST NOT be air bubbles in the tubes Tubes may be cleaned with lint free wipes bubbles may be removed by removing and adding D1 again PLEASE NOTE The ArrayTubes as used in this kit do have a different geometry than the 8 well ArrayStrips that are used in other kits Therefore unlike the directive for ArrayStrips D1 is NOT to be removed from the ArrayTubes before reading 7 Data Acquisition For image uptake you need an ATRO3 reader or an ArrayMate reader respectively Please refer to the reader s manual for image acquisition With the Iconoclust ATRO3 you would be able to further process the images for spot intensity analysis Due to the lack of validated threshold values we currently do not support this kind of analysis for the P aeruginosa test kit however Instead we recommend the data analysis method proposed by Wiehlmann et al 2007 as outlined below An automated version of data analysis will be available for the next generation kit by the end of year 2010 www clondiag com 05 16 04 0004 VO1 14 CLONDIAG 8 Image analysis according to Wiehlmann et al 2007 All images shown below are proprietary of Lutz Wiehlmann Medical School of Hannover Germany The example shown here is the strain P aeruginosa PAK Biotin Control Spots The Arrays contain six spots indicated by arrows in the image that directly bind to Streptavidin Horseradishperoxidase and which have to be positive These spots indicate prop
11. CLONDIAG P aeruginosa Genotyping Kit Array Hybridisation Kit for DNA based assignment of unknown Pseudomonas aeruginosa isolates to known strains Kit order number 205200050 50 reactions ArrayTubes For Investigational Use Only Not Intended for Use in Clinical Diaqnostics CLONDIAG Content PACKOROUND eestasoare osteo artuoata san vormoaatertoanieanses E TENE A E A A EAA AOA AA AEA 3 EE AU a E ET E A A EA AEA S DE E T AEN T EEE EA E T ET ETTE ST 3 GENERAL INSTRUCTIONS FOR USE sccssssscccccsccssssesccccssccssescscessecenssescseessecenseeececessceusseesscesseeeuseesssesseecenseseseeses 4 KIT COMPONENTS STORAGE AND STABILITY sssssecccceecceeeseecccceecsuueeeccceeeeuuueeeecceeessuunseeccseeesauanseeeceeseuaanneeess 6 PROTOCOL areeerates acess E EA EE AE OE E E 8 1 P aeruginosa OINB ioe sisisscsd ca satvsvenw tad canctavavecw saad casavav aicoe totic aiadan avaonsanawabuteddaou aia vanetess Gaon iatawaintess caneialaraumiesaseeesanees 9 2 Linear amplification and biotin labelling 0 0 ccc ccccccessccceseccceesececeesecceeuececeeececeeeeceseecsseeeceseeeeseeees 10 SAVDONI O ssoateciccecsate nu orev dinates see arene E se dds bande ates sts gals wasp rae ua one dna niece nessa ue E TOTO 10 4 Setup of the ArrayMate Reader ccc ccccccccesccccceseccccesecsceuscccceeececuenecsceueecesuecessusecsseeeceseeecssuueecesseceeseeeeess 12 5 Washing after Hybridisation cc
12. RFACE e 2 Washing step after hybridisation add 500 uL of buffer C2 incubate in the thermomixer for 5 min at 30 C 550 rom remove and discard the washing solution e 3 Washing step after hybridisation repeat ane Washing step NOTE a carryover of more than 1 of buffer C1 to the next step will denature the HRP 6 Addition of HRP conjugate NOTE Reagent C3 contains Streptavidin Horseradish Peroxidase HRP that would denature and lose its activity at 60 C Do NOT incubate above 30 C e Combine Reagent C3 HRP Buffer C4 1 100 gt C3 4 the mixture is stable for 1 day at room temperature both reagents are delivered with a surplus of more than 100 each Add 100uL of C3 4 to each tube e Incubate for 10 minutes at 30 C and 550 rpm on a thermomixer e Remove and discard C3 C4 www clondiag com 05 16 04 0004 VO1 13 CLONDIAG e Washing step after binding of conjugate 2x add 500 uL of buffer C5 remove and discard the washing solution WITHOUT TOUCHING THE ARRAY SURFACE Repeat this step once Staining of bound HRP conjugate NOTE acarryover of more than 3 of buffer C3 4 will cause higher background signals donot move ArrayTubes during staining Reagent D1 contains a substrate for Horseradish Peroxidase e Add 100uL of reagent D1 to each tube e Incubate at room temperature WITHOUT agitation for 10 min e Read out without removing D1 CAUTION the tubes MUST be clean underneath the arrays and
13. bottom or across the data matrix barcode This may cause an error label HERE Barcode keep it clean e Avoid contact of data matrix barcode with organic solvents e Avoid touching the bottom of the ArrayTube and keep it clean e Never rinse the tubes with water after hybridisation e Always cap the tubes before incubation in the thermomixer Preparation of the hybridisation mixture e Pre heat the thermomixer to 60 C www clondiag com 05 16 04 0004 VO1 11 CLONDIAG e Add 90 uL of buffer C1 to each labelling product mix gently vigorous mixing results in foaming and put aside Pre washing of the arrays 2 washing steps e Remove the ArrayTube from the bag open the bag at its predetermined breaking point e Add 500 ul of ultrapure water to each tube e Incubate in the thermomixer at 60 C 550 rpm for 2 minutes e Remove and discard the water WITHOUT TOUCHING THE ARRAY SURFACE e Add 200 uL buffer C1 to each tube e Incubate in the thermomixer at 60 C 550 rpm for 2 minutes e Remove and discard buffer C1 e Proceed promptly hybridisation mixtures must be ready when buffer C1 is removed Hybridisation e Transfer each hybridisation mixture 100 uL to a prepared ArrayTube avoid extensive foaming e Incubate for one hour at 60 C and 550 rpm on a thermomixer 4 Setup of the ArrayMate Reader In case you use an ArrayMate reader instead of the ATRO3 reader we recommend to set up the reader after having start
14. c ccecccccsecccceseccceeseccceuscccceececeuecsceusececeueceseeeecsseusecesuuecsseeneceseeeseeees 13 6 Addition of HRP conjugate ccc cccccceseccceseccceescceceecsceesecsceuecceseecssuuseceseuecesseuecesseeeseseuecessunecesaueesesenecs 13 7 Data Acquisition cecasensscnsncisvensoncoienssis lt kessanneadxonossleassiunadensegsacs uuncecuoansdhien muna denaadauiltwsercacnnsdestunwsdenatbideasewestacawaess 14 8 Image analysis according to WiehImann et al 2007 cceccccccceesecccceeeeecceseeesecssseeeesseseeeeceeseeeeseees 15 TROUBLE SHOOTIN O oane E 19 ADDITIONAL INFO RIVIATIO NiesscicotarsascoscsacstousancsanunsaunnsnadanateusancaauaasescetanaaedanndooateusoadedauaanesGounaaantaintansdastanaetousaaasiemecats 20 CONTACT arei A vrs rote se aces uke rete le one ber oer ne les tenia na nec soa AAAA AEAN AAAA E 20 APPENDIX 1 form for 1 level of interpretation the 1 0 string COdE ccccccccceccececeecescscescececcacescscescacecesceeees 21 APPENDIX 2 substances markers marker groups and their respective positions on the array 008 22 APPENDIX 3 Re order numbers of individual COMPONENMS ccecccccessccceesececeesecceeeecceseececeuecseeeeeceeeeeecesees 24 www clondiag com 05 16 04 0004 V01 2 CLONDIAG BACKGROUND CLONDIAG s P aeruginosa Genotyping Kit allows DNA based assignment of unknown P aeruginosa isolates to known strains For Investigational Use Only Not Intended for Use in
15. ed the hybridisation this allows you a restart without time pressure in case of any software problems This is a short instruction only For more detailed information please refer to the ArrayMate User Manual e Switch on the ArrayMate main switch on the rear below the electric cable plug operating switch on the bottom left corner of the front side e Switch on the screen switch right hand below the screen e Login as User R amp D password to be defined by the instrument s administrator on the customer s site and to be initially implemented by the supplier s technical service The user interface is loaded ArrayMate performs internal testing for lt 1 minute e Click on the icon New Run left upper edge of the screen a suggestion for a run name for the new run appears in the top line of the screen www clondiag com 05 16 04 0004 VO1 12 CLONDIAG e You may now modify or change the experiment name at your convenience e Type in your operator ID 5 Washing after Hybridisation Washing after hybridisation 3 washing steps e Remove the ArrayTubes from the thermomixer e Set the thermomixer to 30 C for the following steps cooling will take some minutes e Carefully open the tubes and remove the hybridisation mixture as completely as possible without touching the array surface e 1 Washing step after hybridisation add 500 uL of buffer C2 remove and discard the washing solution WITHOUT TOUCHING THE ARRAY SU
16. er test performance after step 5 Washing after Hybridisation Further the Biotin spots help with the orientation of the image for analysis Spots DNA probes and markers on the image Each DNA molecule probe is represented by two spots on the array A marker may be represented by one or more probes An example P aeruginosa PAK is seen here Marker names cas C 46 C 47 PAGI 3 1 PAGI 3 8 PAGI 2 1 Biotin PAGI 2 3 1 PAGI 2 3 4 PAGI 2 3 5 PAGI 2 3 6 Pf Marker oriC probe PAO probe non PAC each probe is represented by wwo spots www clondiag com 05 16 04 0004 V01 15 CLONDIAG Probe positions on the array groups of markers As outlined above each DNA probe is represented by two spots on the array The array contains 77 different DNA probes no 2 78 in the image below Additionally there is a control probe that contains Biotin number 1 that is printed in hexaplicate position of probes on the array 1 71 71 72 72 73 73 74 74 75 75 76 76 1 A complete list of probe names is to 67 67 68 68 69 69 70 70 O O O O be found in appendix 2 at the end of this document Several probes may contribute to a single marker e g probe oriC PAO and oriC non PAO contribute to marker oriC The markers in turn are grouped into ten different marker groups position of markers on the array legend i ce es ee empty position ae Se a a Biotin Biotin spot group 1 a ae group 2 group 3 P grou
17. he RNA digest yielded sufficient DNA The following procedure is based on the Qiagen DNeasy Blood amp Tissue kit Qiagen order number 69506 CAUTION P aeruginosa is a potential pathogen All procedures prior to complete death of the bacteria need to be performed by properly trained staff in a biosafety Level 2 facility Add bacteria to 180 uL of buffer ATL Amount of bacteria an inoculating loop of 1 uL filled with bacteria this corresponds roughly to 1 8 of a bacterial growth plate overnight culture of bacterial lawn Continue with step 2 of the protocol Purification of Total DNA from Animal Tissues Spin Column Protocol page 29 of the DNeasy Blood amp Tissue Handbook 07 2006 In brief add 20 uL of Proteinase K incubate at 56 C until the tissue is completely lysed After lysis add 4 uL of Qiagen s RNase A solution 100 mg mL to be ordered separately order number 19101 mix and incubate for 2 min at room temperature before continue with step 3 of the Qiagen protocol addition of 200 uL of buffer AL IMPORTANT BEFORE CONTINUING Measure DNA concentration A260 method and check for DNA integrity and absence of RNA agarose gel www clondiag com 05 16 04 0004 V01 CLONDIAG 2 Linear amplification and biotin labelling e Suspend 0 5 2 microgram of clonal and intact P aeruginosa DNA in 5 uL of ultrapure water in a reagent tube suitable for PCR concentrate DNA if necessary by evaporation i
18. he well CAUTION Do not store or handle unused tubes above 60 relative humidity since this may irreversibly corrode the spots e C1 Hybridisation Buffer Store at 18 28 C protect against sunlight Surplus 100 e C2 Washing Buffer 1 Store at 18 C 28 C protect against direct sunlight Surplus 67 e C3 HRP Conjugate 100x Store at 2 8 C protect against direct sunlight Surplus 200 e C4 Conjugate Buffer Store at 18 C to 28 C protect against direct sunlight Surplus 500 e C5 Washing Buffer 2 Store at 18 C to 28 C protect against direct sunlight Surplus 140 e D1 Horseradish Peroxidase Substrate Store at 2 8 C protect against direct sunlight Surplus 140 Expiry date is to be found on each bottle and on the outer package All components have been tested for stability for short term shipment lt 1 week at ambient temperature lt 37 C Please note ArrayTubes B1 and D1 respectively are by far the most expensive test components and hence they are the limiting components of the kit Please keep this in mind when using the kit All components may be ordered separately using the order numbers indicated at the end of this manual For a price list please contact your local representative or our customer service respectively www clondiag com 05 16 04 0004 VO1 6 CLONDIAG Components required but not provided by Clondiag DNA preparation kit The assay has been tested with the DNeasy
19. n a vacuum centrifuge or by precipitation Do not forget to label the vial e Prepare a Master Mix by combining 4 9 uL of B1 2x Labelling Buffer and 0 1 uL of B2 DNA Polymerase per specimen e Add suspended DNA to a 5 ul aliquot of the MasterMix e Perform the following thermocycler protocol required time approximately 3 hours Pre heat cover lid to 105 C 300 sec at 96 C 20 sec at 62 C 50 cycles with 40 sec at 72 C 60 sec at 96 C Cool down to 4 C hold 3 Hybridisation General Precautions e For removal of liquid from the ArrayTubes we recommend the use of plastic Pasteur pipettes with flexible tips Pasteur pipette plastic with a flexible tip C N l flexible tip e Never touch the Array surface Remove liquid with a pipette always place the pipette tip at the cavity between the array and the wall of the tube If you touch the array surface probes may be scratched off and this may cause an error www clondiag com 05 16 04 0004 V01 10 CLONDIAG use the cavity between array and thewall of the tube do nevertouch the array Array e Avoid any complete drying of the array surface during processing i e do not allow it to stay without liquid for more than two minutes e We strongly recommend that the liquid is removed by pipettes instead of inverting the tubes and flicking them out e Always label your ArrayTubes with a laboratory marker at the recommended position Never label them on the
20. onents fails within the expiry date due to other reason than misuse contact CLONDIAG for replacement or refund Terms and conditions apply www clondiag com If you have any problem or question please contact CLONDIAG technical service Quality Control Each batch is stringently tested with the use of standard DNA control material for good performance and correctness of results CONTACT CLONDIAG GmbH Lobstedter Str 103 104 D 07749 Jena Tel 49 0 3641 3111 116 Fax 49 0 3641 3111 120 email dorit lichter clondiag com www clondiag com 05 16 04 0004 V01 20 CLONDIAG APPENDIX 1 form for 1 level of interpretation the 1 0 string code Each group 1 marker exists in a PAO 0 OR in a non PAO 1 version respectively Each group 2 marker like fliC a exists in a positive 1 OR in a negative 0 version respectively This image information is now to be converted into a 0 1 string as outlined below Image and markers version of the marker In this figure there are four image examples for each marker Find out whether your image better resembles the PAO version or the non PAO version respectively and type O or 1 into the string code mask PAO type 0 non PAO type 1 oriC oprL 1 oprL 2 alkB2 citS 1 citS 2 opri 1 opri 2 ampC 1 ampC 3 ampC 4 ampC 5 oe 8088 ei ampC 6 EE ee ampc7 Samu ee bs BI IEI DER Wiis il a l Py j fn positive fliCa
21. p 4 group 5 group 6 group 7 group 8 group 9 group 10 For strain identification only marker group 1 probes 2 33 and marker group 2 probes 34 37 are relevant The other marker groups may be used for the analysis of microevolution within an institution or even within a single patient as outlined by Wiehlmann et al 2007 www clondiag com 05 16 04 0004 V01 16 CLONDIAG 1 level of interpretation the 1 0 string code Each group 1 marker exists in a PAO OR in a non PAO version respectively Each group 2 marker like fliC a exists in a positive OR in a negative version respectively This image information is now to be converted into a 0 1 string as outlined below Image and markers ee eRe ee eee PAGI 2 3 1m PAGI 2 3 4 PAGI 2 3 5 PAGI 2 3 6 Lee version of the marker In this figure there are four image examples for each marker Find out whether your image better resembles the PAO version or the non PAO version respectively and type 0 or 1 into the string code mask see also appendix 1 non PAO type 1 ai EET ee HEHEHE E E h BaT BEER eee ee eee O L S of eo ee F oprL 2 e ee 66 eeee ee alkB2 ee ee coe Dees m eee citS 1 ee ee oe EE EEESE E citS 2 TELNE E oprl 1 oprl 2 ampC 1 ampC 3 ampC 4 eo wa E ee Ob BEREE ampC 5 ampC 6 eee sp eoee ae ee APC positive fliCa negative e E fliCa SNP 1 ane etc pdo fliCa
22. uality we recommend to re check DNA quantity and quality first by loading leftover DNA on an agarose gel e The amount of DNA is crucial A260 readings will cover RNA and other contaminants as well Therefore pure DNA preparation without bulk amounts of RNA are a prerequisite for proper DNA concentration measurement e DNA must be unfragmented as fragmentation reduces the efficiency of amplification and labelling due to the distance between primer and probe binding sites e DNA should be free of RNA as free RNA reduces the efficiency of amplification and labelling by effectively removing primer from the reaction mix due to competitive hybridisation e DNA must be free of any traces of ethanol as ethanol strongly influences the amplification This is an important issue as e g in the top of QIAGEN tubes a drop of ethanol containing washing buffer might get trapped The tubes really need to be centrifuged on high speed Alternatively it is possible to heat the sample prior to adding it to the labelling mix some 5 minutes at 70 C Some problems with samples from the Qiagen EZ1 device for example were resolved after heating the samples e All reagents need to be within the recommended shelf life and stored in the appropriate Way We have good experience with the manual QIAGEN DNeasy kit and the automated device EZ1 Contrarily alkaline lysis mechanical bead beater extraction or ultrasonication in our hands resulted in poor DNA recovery
Download Pdf Manuals
Related Search