SPECIES 2.0
Contents
1. Qchipron USER MANUAL LCD Array Kit SPECIES 2 0 DNA based identification of Animal species Code A 750 04 Code A 750 12 1 Manual SPECIES 2 0 Vers 1 0 2012 N DO oO fF OMN 10 11 12 13 14 15 16 17 Product fOrrmMatiOn eiersiden ta eri E EE A REEE E 3 Li mtended Use ecnernuenaninian a a avian cena eutigede dds 3 1 2 TeSt principles a aa a E a aa aa aaa aaa E aiaa 4 Storage conditions amp kit CONtENE 0 eee eee enee cece ee enee eect canes ee eeaeeeeeeaeeeeeeaaeeeeesaeeeeeeaaeeesenaeeeeeeeetenaeeeeeaas 5 Equipment and reagents not supplied with the kit ccceceeececceeeeeeeeeeeeeeeeeeesecacaeeeeeesesensieeeeeneaees 6 Preparation Ol reageMmts seinne biaevcel vedo E ESA tide ee eat eel dn 6 Warnings and precautions issscicio iaer Sn an A E EEA andes eels adensielen anders 7 General safety information scccccgiedhetieteceets ledges eaeees devas eegaviaciaavidveciefescistledareaws naaaveuteslaeetncag ida Veedeaseet ns 7 Method descriptio Miosina aaa a aaa aaa aaia aaae Ea aa aE i A aaa 8 TA POR Seniora a oa E R A aS E EAE EN 8 7 2 Hybridisation Detection eesseeeeseesrreeeerrecernreseerernearrnnederanaetanestenuatrnnaatdaaaaetnadtenaddtnnneddaaaataaaeennas 8 Array description 2 2 cece ee eene eee e tenn cette ee ee tee ea aeee seca aeeeeeeaeee ENEE EEEREN EAEE EEEE EREA EEEE 9 PROLOCOD aaa hea skbhaecds aed ended ecu eanen a a aeeededs 10 9 1 POCRampiticatio Mron a Ga cancdeanabads a a a a2. Observation Probable causes Solution Observation Probable causes No signals detected including guide dots Wrong reaction set up wrong temperature use of wrong hybridisation buffer Compare the experimental set up with the given protocol check temperature of the water bath compare the letters printed on the hybridisation buffer label and the chip label should be B Guide dots detected but no other signals visible PCR failure DNA extraction inefficient template DNA degraded DNA content too low or test result for target organisms is negative Use fresh PCR chemicals make sure to run the controls provided with kit check the PCR cycler program try an alternative DNA extraction procedure Signals detected but background is too high Wrong reaction set up or incubation times too long 18 Manual SPECIES 2 0 Vers 1 0 2012 14 Solution Observation Probable causes Solution Observation Probable causes Solution Observation Probable causes Solution Observation Probable causes Solution Make sure the Modulator has been added at all stages according to the protocol monitor incubation times and temperatures for hybridisation labelling and staining carefully Bleeding guide dots and very strong control signals Staining or secondary labeling time is too long Reduce the incubation time gradually in 1 minute steps Lots of small dark particles on chip surface 1 x wash buffer is
3. contact your local distributor or the Chipron support team to learn more about the automatic analysis options with the SlideReader software Device performance All DNA sequences underlying the design of primer and probes have been carefully chosen and thoroughly confirmed by broad comparisons in publicly available databases BLAST analysis No indication for cross reactivity apart from those as described below the specificity table on page 9 could be revealed by such n silico analysis It should be noted that as for all PCR and hybridisation based molecular tests it can not be excluded that rare genotypes comprising variations within the sequence regions used for the assay design may remain undetected Additionally other yet unknown yet unpublished species especially among the Bovidae might contain highly similar sequence regions in their genomes and could therefore interfere with the assay performance The assay in its present form reflects the current state of knowledge of Chipron GmbH Sensitivity The sensitivity of the assay is mainly determined by the number of PCR cycles and the binding affinity of the capture probes during hybridisation Under the given protocol conditions hybridisation buffer temperature and duration the binding affinity of the capture probes is well standardised and allows for the detection of 10 fmol of single stranded biotinylated target sequences The number of PCR cycles on the other hand can ea
4. individual targets or equimolar mixtures thereof In unequimolar mixtures of multiple target sequences the detection limit for targets of lower concentration will be raised by competition with targets of higher concentration For DNA detection assays based on multiplex PCR amplification like the SPECIES 2 0 Kit this implies that the DNA concentration of the most prominent species in a sample determines the experimental detection limits for species of lower abundance A typical dynamic range of Multiplex amplifications with respect to the targets of the highest and the lowest abundance overspans 3 4 logs e g 105 102 For the analysis of meat preparations with the SPECIES 2 0 LCD Array in samples consisting of meat from mainly one species 99 additions of other meat species in the range of 0 1 should be detectable An experimental proof has been provided by Bush and colleagues 1 Determination of device performance The type of Taq polymerase and the Theromcycler instrument used can have a strong influence on the assay performance Therefore all performance tests and assay evaluations have been undertaken with various combinations of the following instruments and enzyme preparations No significant differences in the test performance have been observed with any combination of the instruments and enzymes listed below Preparations of Taq polymerase AmpliTaq Gold PCR Master Mix Applied Biosystems GmbH Code 4318739 HotStar Ta
5. precipitation Manual SPECIES 2 0 Vers 1 0 2012 Storage conditions amp kit content When stored at the indicated temperature and not handled otherwise as specifically described all components are stable until their expiry date Expire dates are printed on the components label or the labels of the surrounding packages More than 6 freezing and thawing cycles should be avoided for components which require frozen storage Prepare aliquots if necessary Component A 300 04 32 tests A 300 12 96 tests Storage LCD Arrays 1 box 04 chips 3 boxes 12 chips 4 C to 28 Wash Powder 1 bottle 2 bottles 4 C to 28 Primer MEAT Primer Mix 1x 100 ul 2x 100 ul 10 C to 25 C Detection Kit Modulator 1 x 300 ul 2 x 300 ul 4 C to 28 Dilution Buffer 1 x 2000 ul 2 x 2000 ul 4 C to 28 Stain 1 x 2000 ul 2 x 2000 ul 4 C to 28 Detection Kit II Hybridisation Buffer 1x 1750 ul 2 x 1750 ul 10 C to 25 C Label 1x 20 ul 2 x 20 ul 10 C to 25 C Additional supplies Analysis Matrix a Manual Pattern File MSDS 1CD 1CD Chip Box Connectors 2 connectors 6 connectors AN Keep the Stain solution always dark protected from direct light 5 Manual SPECIES 2 0 Vers 1 0 2012 Equipment and reagents not supplied with the kit Instrumentation Thermal cycler Water bath Centrifuge for LCD Arrays Micro pipettes optional SlideScanner PF3650u SlideReader Analysis Software Reagents amp Materia
6. 5 ul 270 ul Modulator 3 0 ul 15 ul 30 ul Label 0 2 ul 1ul 2 ul Total 30 2 ul 151 ul 302 ul Mix well by vortex or intensive pipetting 9 Apply 28 ul of the label mix to each field of the slide Make sure that the label mix has contact with the entire reaction zone of the respective array field avoid any contact of the pipette tip with the chip surface 10 Incubate the slide at room temperature for 5 min 11 Replace the wash buffer in all three containers and repeat the wash procedure from step 7 12 Dry the slide as in step 8 9 2 3 Staining 13 Apply 28 ul of Stain solution to each field and incubate for 5 minutes at room temperature Make sure that the fields do not contain any traces of washing solution wait until fields are completely dry A Avoid any contamination of the Stain stock solution Prepare aliquots if necessary 14 Stop the staining process by rinsing the slide in the last wash container from step 11 for 15 seconds 15 Dry the slide as in step 8 The slides are now ready for read out and can be kept in the dark for several years without a significant loss of signal intensity 13 Manual SPECIES 2 0 Vers 1 0 2012 9 3 Short protocol 1 Set water bath temperature to 35 C For each reaction mix 22 ul Hybridisation buffer g 2 ul of Modulator and 10 ul of PCR product Apply 28 ul of this solution to the respective array field Incubate the slide at 35 C for 30 minutes in humid
7. Combine the PCR product with the Hybridisation Mix Add 10 ul of the PCR product to the respective aliquot of Hybridisation Mix Mix well by pipetting up and down for several times do not vortex Initiate the hybridisation Place the slide in the humidity chamber and pipette 28 ul of the PCR Hybridisation mixes to the respective fields Make sure that the PCR Hybridisation mixes come into contact with the entire reaction zone of the respective array field avoid any contact of the pipetting tip with the chip surface Transfer the closed humidity chamber as quickly as possible to the preset water bath the chamber will float on the surface Incubate the slide at 35 C for 30 minutes Prepare three wash containers with 1 x wash buffer 150 ml each Prepare the 1 x wash buffer from the 20 x wash buffer concentrate by adding 50 ml of concentrate to 950 ml of deionized water Mix thoroughly Submerge the slide completely in the first container with the wash buffer and move it 3 times slowly backward and forward Submerge the slide quickly to avoid field to field cross talk Repeat the procedure in wash container 2 Subsequently transfer the slide to container 3 and incubate for 1 minute Dry the slide by spinning for 15 seconds in the CHIP Spin FVL2400N Cat No HS 500 01 12 Manual SPECIES 2 0 Vers 1 0 2012 9 2 2 Labelling 8 Prepare the labelling mix Number of reactions 1 4 setup 5 8 setup 10 Dilution Buffer 27 0 ul 13
8. a aaa 10 911 PCR Set pencrs asiaan aaa dies id a a iting ideaa deea 10 QUA Template ic aicetaeccacacsesadevchaacceectcqdaetehaanscuaacadedantinancdedecuanacedtecas cacaaadaden a a a aaa aada 10 IL Cycler SCUINGS eenaa EA EREE 11 9 1 4 Agarose gel examination 0 0 0 cette ee eeeeeeeeeeeeeee eee ttun tt ttunttuna Ennan EE nant E naat nn nane nn nanen nanna 11 9 2 LCD Array hybridisation and detection eeccceeceeceeceeeenneeeeeeeeeeeeaeeeeeeaeeeeeaeeeseenaeeeennaeeeeeeaas 11 JAN TAYDrICISAUO Ms vovse ces a vaneceeemereestsncdie baebsaleads vaneden veut ces caanggs niadaddeyetedeeadaceesalieesest 12 9 22 LabellinQiis cccisccccceiseccceiiscceeves secavessccceees ieee sieewesscccevesccaevessecuevesseaeuusacceeeeaccedeessteceusaced eased 13 9 2 3 AMN G e n E Meindl asl dine een lebie adel caine el 13 9 3 SOM Protocolar sana aa ceacenepcaaansatsanecadvseduawusa ced suka adauas exedaat eawncaduaverde lean tanereats 14 Analysis and interpretation of the results 0 c cee ccccceceeeeeneeeeeeeeeeeeeaeeeeeeaeeeeeeaaeeeeeeaeeeeesaeeesnnaeeeeeeeseeaaes 15 10 1 General remarks 000 0 eceeeeeeeeeeeeeee eter teeeee eee eeeeeaeeeeeeeaeeseeeaeeeseeaaeeseeaaeeesecaeeeseeaeeeeeeeeeeeeeeseaes 15 10 2 Methodsof analySis is ciieciecntetacdesvacaetvsdeccee A A deiads 15 10 2 1 Analysis by simple optical examination with naked eye eecceeeeeeeeeeeeeeeeeeeneeeeeeaes 15 10 2 2 Analysis by transmission light scanning and SlideReader sof
9. g Plus Master Mix Qiagen GmbH Code 203645 PCR Thermocycler Mastercycler Eppendorf AG Code 5333 000 018 TProfessional Standard Biometra Analytik Jena AG Code 070 951 labcycler Basic SensoQuest GmbH Code 011 103 AmpliTaq Gold is a registeredtrade mark of Roche Molecular Systems Inc MasterCycler is a registeredtrade mark of Eppendorf AG 17 Manual SPECIES 2 0 Vers 1 0 2012 12 13 Device limits The following factors might limit the assay performance total concentration of extracted DNA is too low or DNA is degraded PCR amplification is hampered by PCR inhibitors which have been co extracted use of PCR additives which can interfere with the hybridisation avoid additives like Urea DMSO Betaine etc use of non calibrated or functionally impaired instruments pipettes water bath PCR cycler any variation from the protocol given in this user instruction Trouble shooting General remarks In cases where unexpected results are obtained or when signal intensities or background levels appear to be different from usual observations check the following list first kit components and PCR chemicals were not expired agarose gel examination of PCR products is positive correct results with positive and negative controls correct combination of primer mixes chip types hybridisation buffer and protocol Detailed trouble shooting Observation Probable causes Solution
10. hole procedure will take 3 4 hrs depending on the duration of the PCR amplification 8 Manual SPECIES 2 0 Vers 1 0 2012 Array description Each LCD Chip contains eight identical arrays in rectangular reaction chambers which can be addressed individually Functional controls to monitor hybridisation secondary labeling and staining are located in three corners The arrays of the SPECIES 2 0 kit consist of an 8 x 8 pattern with average spot diameters of 350 um The species specific capture probes are positioned as vertical duplicates q 2 3 4 JEJEJE E y ETE VE 8 8 7 8 SPECIES 2 0 here 0000000 E 0377 001 001 OOoododcd GOS oh lt Name Hyb Ctrl Beef 1 Buffalo Pork Sheep Goat Horse Donkey 2 Rabbit Hare Chicken Turkey Goose No Species Name Hybridization Control Bos taurus Mall Duck Bubalus bubalis Musc Duck Sus scrofa Dog 3 Ovis aries Cat 4 Capra hircus Kangaroo Equus caballus Ostrich Equus asinus Bison Oryctolagus cuniculus Red Deer Lepus europaeus Fallow Deer Gallus gallus Roe Deer Meleagris gallopavo Camel 5 Ansa albifrons Seagull Cross reactivity of the capture probe for Beef with 100 Bison may occur Cross reactivity of the capture probe for Donkey with 100 Horse may occur Capture probe detects Canis lupus C lupus familiaris C lupus chanco and Canis indica Capture probe detects several species f
11. horeses 2 0 agarose gel LCD Array hybridisation and detection Detailed protocol for first time user advanced users refer to short protocol chapter 9 3 General Remarks Since the working principle of LCD Arrays is DNA DNA hybridisation the specificity and sensitivity of the assays are mainly controlled by the hybridisation stringency during the 30 minute hybridisation step Apart from the concentration of the interacting molecules the two factors with the biggest influence on hybridisation stringency are temperature and buffer composition e g concentration of salts formamide urea etc It is crucial that the temperature during hybridisation and the pipetting volumes are precisely controlled The use of calibrated thermometers and micro pipettes is strongly recommended Deviations of more than 1 C or 1 ul can have severe effects 11 Manual SPECIES 2 0 Vers 1 0 2012 9 2 1 Hybridisation 1 Set water bath temperature to 35 C and add one droplet of water to each corner of the humidity chamber Do NOT preheat the LCD Chip The chip should be equilibrated to room temperature Prepare the Hybridisation Mix Make sure that all components are equilibrated to room temperature Number of reactions 1 4 set up 5 8 set up 9 Hybridisation buffer 22 ul 110 ul 198 ul Modulator 2 ul 10 ul 18 ul Aliquot per 24 ul 24 ul 0 2 ml reaction vials or 8 well PCR strips are well suited for setting up the reactions
12. ise Presence and absence in this respect is defined by the assay specific detection range and limits for each parameter see chapter 11 Device performance 11 1 Sensitivity Although different signal intensities can be observed during the analysis of LCD Arrays and these intensities are generally correlated with the amount of target copies in the starting material it should be noted that LCD Arrays have not been validated as tools for absolute quantification Methods of analysis The formation of dark visible precipitates at positions spots where DNA DNA hybridisation between the PCR amplicons and the immobilized capture probes took place combined with the crystal clear polymer support LCD chip offers the opportunity to use two different analysis methods Simple optical examination naked eye with or without magnifying lenses or transmission light scanning followed by software assisted image analysis details are given below The experiments of all validations and performance tests of the product have been analysed with both methods in parallel with identical results Regardless of the method chosen for analysis negative controls should always be included into the experimental scheme to ensure that no artificial background signals are detected due to cross contamination or over staining see chapter 13 Trouble shooting Analysis by simple optical examination with naked eye Each LCD Array Kit contains a patter
13. ity chamber Prepare 3 wash containers with 150 ml each of 1x wash solution 1 x wash buffer freshly prepared from 20x concentrate Rinse slide in wash container 1 and 2 for 10 seconds each and incubate in wash container 3 for 1 minute Dry the slide by spinning it for 15 seconds in the CHIP Spin Centrifuge Prepare the labelling mix by combining 27 ul of Dilution buffer 3 ul of Modulator and 0 2 ul of Label per array field Apply 28 ul of label mix to each field of the slide and incubate for 5 minutes at room temperature Replace the wash solution in all containers and repeat the wash procedure from step 5 Dry the slide as in step 6 Apply 28 ul of Stain solution to each field of the slide and incubate for 5 minutes at room temperature Avoid contamination of the STAIN solution with residues of Label solution Stop the staining after 5 minutes by rinsing the slide in the last wash container from step 9 for 10 seconds Dry the slide as in step 6 the slide is now ready for analysis 14 Manual SPECIES 2 0 Vers 1 0 2012 10 10 1 10 2 10 2 1 10 2 2 Analysis and interpretation of the results General remarks LCD Arrays generate qualitative results indicating the presence or absence of the respective parameter species within the sample material used for DNA extraction and PCR amplification Since the assay principle is based on DNA detection vital and dead organisms will be identified likew
14. l 7 5 ul 13 5 yl Taq Polymerase 5 U ul 0 3 ul 1 5 ul 2 7 ul PCR grade water 14 7 ul 73 5 ul 132 3 pl Total volume 20 0 ul 100 0 ul 180 0 pl Aliquot into 4x 20 0 ul 8 x 20 0 ul 9 1 2 Template Add 5 0 ul of extracted DNA as template to each reaction tube Note If larger or smaller volumes of template need to be added reduce the amount of added water accordingly 10 Manual SPECIES 2 0 Vers 1 0 2012 9 1 4 9 2 Cycler settings The cycle regime given below has been optimised for the use of HotStarTaq Plus Master Mix Qiagen GmbH Code 203645 TProfessional Standard Cycler Analytik Jena AG Code 070 951 When other combinations of PCR cycler and enzyme will be used slight modifications of the given protocol could become necessary Please contact your local distributor or the Chipron support team if you wish assistance for any kind of assay optimization Step Duration Temperature Ramp Note 1 Initial denaturation 5 00 min 95 C longer with some Hot Start enzymes 2 35 repetitions of Denaturation 0 30 min 94 C 3 C sec Annealing 0 45 min 57 C 3 C sec Elongation 0 45 min 72 C 3 C sec 3 Strand completion 2 00 min 72 C Increase cycle number for higher sensitivity or decrease cycle number for lower sensitivity Agarose gel examination When the arrays are used for the first time or optimisation becomes necessary it is recommended to analyse the PCR amplification by agarose gel electrop
15. ls Reagents for DNA extraction PCR chemicals PCR grade water Deionised water Disposable gloves Sterile filter tips PCR reaction vessels 3 wash containers 1l bottles Preparation of reagents Wash Buffer To prepare the 20 x wash buffer concentrate dissolve the content of one wash powder bottle in 1 liter adjustable to 35 C cat no HS 500 01 Chipron GmbH range from 2 ul to 1000 yl cat no HS 300 01 Chipron GmbH cat no HS 200 01 Chipron GmbH Taq Polymerase Buffer dNTPs 150 to 400 ml each of deionised water To prepare the 1 x wash buffer add 50 ml of 20 x wash buffer concentrate to 950 ml of deionised water Prepare the 1 x wash buffer always fresh Stability of Wash Buffer solutions 20 x Concentrate 1 x Working solution 2 month at room temperature 12 month at 4 C If formation of precipitate occurs warm the solution to 42 C and let equilibrate to room temperature prior to use min 10 days at room temperature Manual SPECIES 2 0 Vers 1 0 2012 Warnings and precautions All steps described in the protocol should only be carried out by well trained lab personnel Read the manual carefully and completely before starting Avoid any exposure to light of the Stain solution Always keep it dark All reagents in the kit are optimised for this particular test Substitution of kit reagents may effect the performance The same general safety guidelines which app
16. ly to the sample material should be followed during the whole protocol The PCR products generated in the first protocol step have to be considered as contamination sources for further PCRs Therefore all hybridisation washing staining drying and analysis steps should be carried out in the post PCR area Observe the standard guidelines for working in a PCR molecular diagnostic laboratory to prevent contaminations General safety information When working with chemicals make sure that you always wear a suitable lab coat disposable gloves and protective goggles For more information about our products please refer to the appropriate material safety data sheets MSDS which can be found on the CD provided with the kit Hybridisation Buffer contains formamide gt 50 and N Dodecanoyl N methylglycin Sodium salt Hybridisation buffer should therefore be handled as formamide Toxic harmful irritant Risk and safety phrases R61 R36 R38 24 S26 Wash Powder contains N Dodecanoyl N methylglycin Sodium salt Harmful irritant Risk and safety phrases R36 S24 S26 Stain contains 3 3 5 5 Tetramethylbenzidin gt 0 5 Harmful irritant Risk and safety phrases R20 21 22 R36 37 38 R40 Self Assessment Modulator Harmful irritant Risk and safety phrases R36 37 38 S26 36 Self Assessment 7 Manual SPECIES 2 0 Vers 1 0 2012 7 1 7 2 Method description The test consists of two main
17. n graphic with the imprint of an array specific Analysis Matrix for simple data analysis The Analysis Matrix displays the array pattern for the specific array type a table with the spot number code and the respective spot identities Genera or species A simple comparison of all visible spots on the array with the corresponding positions given in the Analysis Matrix will lead to the correct data interpretation Analysis by transmission light scanning and SlideReader software An alternative method for data analysis including the generation of comprehensive data reports in PDF format is the use of the SlideScanner PF3650u in combination with the SlideReader Software Product Order No Company SlideScanner PF3650u HS 300 01 Chipron GmbH Berlin Germany SlideReader Software HS 200 01 Chipron GmbH Berlin Germany 15 Manual SPECIES 2 0 Vers 1 0 2012 11 Detailed instructions for the use of SlideScanner and SlideReader software are given in the SlideReader Manual The pattern file for automatic analysis with the SlideReader software is provided as data file on the CD which is part of every LCD Array kit Choose the directory Pattern File and dependent on the SlideReader software version in use one of the two following files SlideReader Vers 6 to 9 use the file Gal 00377 SPECIES 2 0 txt SlideReader Version 11 and higher use the file 0377 SPECIES 2 0 gal Required software settings Cut off set to 2000 Please
18. opavo Ansa albifrons Mallard Duck Muscovy Duck Dog Cat Kangaroo Ostrich Bison Red Deer Fallow Deer Roe Deer Camel Seagull Anas platyrhyncos Cairina moschata Canis lupus familiaris Felis catus M giganteus M rufus Struthio camelus Bos bison Cervus elaphus Dama dama Capreolus capreolus Camelus sp Larus sp List of species which can be detected by hybridisation of specific capture probes Manual SPECIES 2 0 Vers 1 0 2012 1 2 Test principle Using extracted DNA as starting material 1 small fragments of the mitochondrial 16S rRNA genes of the target organisms will be amplified by the initial PCR During this amplification the generated PCR fragments are labelled with Biotin 2 PCR amplicons are hybridised to specific capture probes on the surface of the array 3 Non specifically bound PCR amplicons will be removed by short washes under high stringency The remaining specifically bound amplicons can be visualized by incubation with a Streptavidin Peroxidase conjugate and the subsequent formation of a dark precipitate after incubation with peroxidase substrate TMB provided as staining solution 4 Y K d mo ee a S ae extracted PCR with biotin DNA incorporation Figure 1 Test principle G Array hybridisation PCR product probe TMB substrate J me s o F precipitate PCR product yd a capture m probe E NY bd Detection by TMB
19. procedure steps PCR amplification of DNA fragments with biotin incorporation Hybridisation of PCR fragments to LCD Arrays and detection PCR One primer mix is provided with the kit The multiple primer pairs in this mix target a portion of the 16S RNA gene of a broad range of Vertebrate species When using the primer mix MEAT distinct amplification products of 115 125 bp can be expected species dependent Examples of fragment sizes Species Size Bos taurus 116 bp Sus scrofa 119 bp Equus caballus 124 bp Gallus gallus 121 bp Struthio camelus 122 bp Hybridisation Detection The labelled PCR fragments are combined with the hybridisation buffer provided and hybridised to the individual array fields of one chip During hybridisation the labelled PCR fragments will bind to the specific capture probes immobilized as spots on the bottom of each field Following a short washing procedure each field is incubated with a secondary label solution enzyme conjugate After a second washing step the positions spots where PCR fragments and secondary label are bound can be visualized as blue precipitate formed by the enzyme substrate provided as STAIN The data read out can either be done by simple naked eyed examination using the Analysis Matrix provided with the kit or alternatively with the scanner and software from the Analysis Package which can be obtained from Chipron GmbH For experienced users the w
20. rom the subfamily Felinae Felis Puma Leopardus Lynx Capture probe detects both species of the Genus Camelus C bactrianus amp C dromedarius Capture probe detects several species from the Genus Larus L argentatus L cachinnans L occidentalis L dominicanus L glaucoides 1 i a v 4 i u a E N AN g F a S 3 H Na 2 i Pd pr ed a i 0000000 0 ODOCOCOOOOO OCOOOOOO 00000000 og Species Anas platyrhyncos Cairina moschata Canis lupus familiaris Felis catus M giganteus M rufus Struthio camelus Bos bison Cervus elaphus Dama dama Capreolus capreolus Camelus sp Larus sp 9 Manual SPECIES 2 0 Vers 1 0 2012 9 1 9 1 1 Protocol PCR amplification PCR set up We recommend the HotStarTaq Plus Master Mix Kit Qiagen Code 203645 Using a 2 x PCR Master Mix incl Taq Polymerase dNTPs Buffer and MgCl Number of reactions 25 ul each 1 4 set up 5 8 set up 9 2x Master Mix incl MgClz final 1 5 2 0 mM 12 5 ul 62 5 ul 112 5 ul Primer Mix MEAT 1 5 ul 7 5 ul 13 5 ul PCR grade water 6 0 ul 30 0 ul 54 0 ul Total volume 20 0 ul 100 0 ul 180 0 ul Aliqout into 4x 20 0 ul 8 x 20 0 ul Using Taq Polymerase dNTPs and Buffer as separate components Number of reactions 25 ul each 1 4 set up 5 8 set up 9 10x PCR Buffer incl 15 20 mM MgCl2 2 5 ul 12 5 ul 22 5 ul dNTP mix 10mM each 1 0 ul 5 0 ul 9 0 ul Primer Mix MEAT 1 5 u
21. s Product Description Order No Slide Reader Software 1 License HS 200 01 Scanner PF 3650 Slide Scanner w 10um resolution HS 300 01 CHIP Spin FVL 2400N Mini Centrifuge for LCD Arrays HS 500 01 Contact manufacturer Chipron GmbH Tel 49 0 30 787994 70 Eresburgstrasse 22 23 Fax 49 0 30 787994 99 D 12103 Berlin Email info chipron com Germany Support support chipron com Symbol key The following symbols are used on labels of the kit box and components therein ttal Manufacturer z Number of tests Lot number d Date of expiry month year Order number 4 Storage temperature range C 20 Manual SPECIES 2 0 Vers 1 0 2012
22. sily be adjusted to modulate the sensitivity range according to the experimental demands The maximal sensitivity of the SPECIES 2 0 LCD Array kit for 16S rRNA sequences of the target organisms represented by specific capture probes is approx 0 1 Genome Equivalent GE ul and will be reached with 42 PCR cycles Note Detection limits below 1 GE reaction are not unusual for mitochondrial target genes like the 16S rRNA gene which are present in high copy numbers cell tissue dependent 16 Manual SPECIES 2 0 Vers 1 0 2012 The assay sensitivity has been measured with serial dilutions of standardized genomic DNA extracts or calibrated plasmid solutions containing fragments of the 16S rRNA genes Example Bos Taurus Genome size 2 65 x 10 base pairs gt 3ng equal 1x103 GE gt 3pg equal 1GE For most users of the SPECIES 2 0 LCD Array kit the relative detectable amounts of species DNA in mixed meat preparations will be more interesting than the absolute sensitivity It should be noted that due to competition the simultaneous amplification of several different target sequences in one PCR reaction Multiplex PCR will constrict the dynamic range of the assay and the detection limits for the individual targets The bigger the difference in the starting concentration of two targets the stronger the effects of competition for the target of lower concentration The sensitivity values given above in GE ul are valid for amplifications of
23. too old formation of precipitates Prepare a fresh solution of 1 x wash buffer from 20 x concentrate Negative control shows species specific signals Contamination of PCR chemicals or primer mixes or sample carryover during the hybridisation protocol Use fresh PCR chemicals make sure to follow the general guide lines for PCR set up to avoid laboratory born contaminations If necessary use new primer mixes Make sure that no well to well cross talk occurs during the hybridisation steps Only single spots are seen for some features not duplicates The missing spots have been mechanically removed during the pipetting or drying steps or by touching the surface of the reaction chambers Use solely the Chip Spin FVL2400N centrifuge for drying steps Higher g forces can result in spot dislocations Avoid any contact with the reaction zone surface of the array fields If none of the above given solutions solves your problem please get in contact with your local distributor or contact our support team at Literature support chipron com 1 Iwobi A Huber I Hauner G Miller A and Busch U 2011 Biochip Technology for the Detection of Animal Species in Meat Products Food Analytical Methods Vol 4 No 3 pp 389 398 19 Manual SPECIES 2 0 Vers 1 0 2012 15 16 17 Order information Product Quantity Order No SPECIES 2 0 32 Tests 04 Chips A 750 04 SPECIES 2 0 96 Tests 12 Chips A 750 12 Related Product
24. tware e 15 DEVICE performance sicions a Naa E aaia E daaa aa a a a aaa 16 VARA SOMSIIVILY cae A saadeus saadedenaatededivvanateeeezsndss 16 11 2 Determination of device performance ccccececcece cee ce cece eeeeceaeeeeeeeeseceaeaeeeeeesecsnaeeeeeeeeeenaees 17 Device IMIE fess ceccy acces estes detec cate ate a E EE AE EE ET N EEEE EEA feces ites 18 Trouble SMOOUIMNG essnpn a a E a A aleeee rears 18 MOTTON asispa E S E E E E E E eden 19 Order informato Mesna EEEE EE R 20 Contact manufacturer andene eranen EENEN 20 SVIMDOW KEY asra A E E montane aie eaveit aeivene ade 20 2 Manual SPECIES 2 0 Vers 1 0 2012 Instructions for use 1 1 1 Instructions version SPECIES 2 0 V 1 0 2012 ENG Revised 26 July 2012 Product information Product name LCD Array Kit SPECIES 2 0 Order code Kit size A 750 04 32 Tests 04 Chips A 750 12 96 Tests 12 Chips Intended use This array has been developed for rapid easy and reliable identification of animal DNA in fresh meat preparations and products manufactured thereof DNA of animal species most commonly used in food production will be detected in parallel using extracted DNA as starting material Beef Buffalo Pork Sheep Goat Horse Donkey Rabbit Hare Chicken Turkey Goose Bos taurus Bubalus bubalis Sus scrofa Ovis aries Capra hircus Equus caballus Equus asinus Oryctolagus cuniculus Lepus europaeus Gallus gallus Meleagris gall
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