Milk ID 2.0
Contents
1. positioned as vertical duplicates The positive detection of each species is indicated by two circular hybridization signals Functional controls to monitor hybridisation secondary labeling and staining are located in three corners The arrays of the Milk ID 2 0 kit are 7 x 7 patterns with average spot diameters of 350 um Milk ID 2 0 CsNr 0396 d 1 J J Specificity Hyb Ctrl jue Cow Bos taurus 1 Ovis aries Capra hircus Buffalo Bubalus bubalis Camel Camelus sp Equus caballus Donkey ON Em 2 3 4 5 6 7 8 9 i Homo sapiens s Capture probe detects both species of the Genus Camelus C bactrianus amp C dromedarius Cross reactivity of the capture probe for Donkey with 100 Horse may occur Figure2 Array Pattern and probe specificities Manual Milk ID 2 0 Vers 1 0 2013 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 accompanying packages More than 6 freezing and thawing cycles should be avoided for components which require frozen storage Prepare aliquots if necessary Component A 850 04 32 tests A 850 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 Mix MEAT 1x 75 u2. 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 Manual Milk ID 2 0 Vers 1 0 2013 7 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 B 22 ul 110 ul 198 ul Modulator 2 Ml 10 ul 18 yul Aliguot per 24 ul 24 ul 0 2 ml reaction vials or 8 well PCR strips are well suited for setting up the reactions 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
3. C chipron USER MANUAL LCD Array Kit Milk ID 2 0 Customized LCD Array Kit Code A 850 12 Manual Milk ID 2 0 Vers 1 0 2013 1 Introduction This array has been developed for rapid easy and reliable identification of animal DNA in milk and milk products Animal DNA of species most commonly used in milk production like cow sheep goat buffalo horse and camel will be detected in parallel The assay is well suited for the analysis of milk and milk product preparations containing milk from more than one animal species 1 1 Method Using extracted DNA extracted from milk or milk products as starting material 1 small fragments of the mitochondrial 16S rRNA genes will be amplified by the initial PCR During this amplification the generated PCR fragments are labelled with Biotin 2 PCR amplicons are hybridised with species 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 TMB substrate PCR PCR recipitate Bo 2 product p P i product 3 Pe i 1 ah E l af 4s 1 e capture ef capture 5 probe 3 probe 23 mand TP extracted PCR with biotin Array De
4. 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 Manual Milk ID 2 0 Vers 1 0 2013 7 2 2 Labelling 9 Prepare the labelling mix Number of reactions 1 4 setup 5 8 setup 10 Dilution Buffer 27 0 ul 135 ul 270 ul Modulator 3 0 ul 15 ul 30 ul Label 0 2 ul 1 ul 2 ul Total 30 2 ul 151 ul 302 ul Mix well by vortex or intensive pipetting 10 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 11 Incubate the slide at room temperature for 5 min 12 Replace the wash buffer in all three contai
5. ield 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 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 Manual Milk ID 2 0 Vers 1 0 2013 10 Order information Product Milk ID 2 0 Milk ID 2 0 Related Products Product Slide Reader Software Scanner PF 7250u CHIP Spin FVL 2400N Contact manufacturer Symbol key Quantity Order No 32 Tests 04 Chips A 850 04 96 Tests 12 Chips A 850 12 Description Order No 1 License HS 200 01 Slide Scanner w 10um resolution HS 300 01 Mini Centrifuge for LCD Arrays HS 500 01 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 The following symbols are used on labels of the kit box and components therein N Number of tests sal Manufacturer LOT Lot number Order number 2 Date of expiry month year 4 Storage temperature range C Manual Milk ID 2 0 Ver
6. l 2x 75 ul 10 C to 25C Detection Kit I 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 44 Cto 28 Detection Kit II Hybridisation Buffer B 1 x 1750 ul 2x 1750 ul 10 C to 25 C Label 1x25 ul 2x25 ul 10 C to 25 C Additional supplies Manual Pattern File MSDS 1 CD 1 CD oe Chip Box Connectors 2 connectors 6 connectors AN Keep the Stain solution always dark protected from direct Light Manual Milk ID 2 0 Vers 1 0 2013 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 Materials Reagents for DNA extraction PCR chemicals PCR grade water Deionised water Disposable gloves Sterile filter tips PCR reaction vessels 3 wash containers 1I bottles Preparation of reagents WashBuffer adjustable to 35 C cat no HS 500 01 Chipron GmbH range from 2 ul to 1000 ul cat no HS 300 01 Chipron GmbH cat no HS 200 01 Chipron GmbH Taq Polymerase Buffer dNTPs 150 to 400 ml each To prepare the 20 x wash buffer concentrate dissolve the content of one wash powder boitle in 1liter of deionised water To prepare the 1 x wash buffer add 50 ml of 20 x wash buffer concentrate to 950 ml of dei
7. l 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 Riskandsafetyphrases R61 R36 R38 S24 S26 Wash Powder contains N Dodecanoyl N methylglycin Sodium salt Harmful irritant Riskandsafetyphrases R36 S24 S26 stain contains 3 3 5 5 Tetramethylbenzidin gt 0 5 Harmful irritant Riskandsafetyphrases R20 21 22 R36 37 38 R40 Modulator Harmful irritant Riskandsafetyphrases R36 37 38 S26 36 Self Assessment Self Assessment Manual Milk ID 2 0 Vers 1 0 2013 7 Protocol 7 1 PCR amplification 7 1 1 PCR set up Using a 2 x PCR Master Mix incl Tag Polymerase dNTPs Buffer and MgClz Number of reactions 25 ul each 2x Master Mix incl MgCle final 1 5 2 5 mM Primer Mix Meat PCR grade water Total volume Aligout into 7 1 2 Template 12 5 ul 1 5 ul 6 0 ul 20 0 ul 4 set up 5 8 set up 9 62 5 ul 112 5 ul 7 5 ul 13 5 ul 30 0 ul 54 0 ul 100 0 ul 180 0 ul 4x 20 0 ul 8 x 20 0 ul 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 Manual Milk ID 2 0 Vers 1 0 2013 7 2 Cycler settings The cycle regime given below has been o
8. ners and repeat the wash procedure from step 7 13 Dry the slide as in step 8 1 2 3 Staining 14 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 15 Stop the staining process by rinsing the slide in the last wash container from step 12 for 15 seconds 16 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 Manual Milk ID 2 0 Vers 1 0 2013 7 3 Short protocol 10 11 12 13 Set water bath temperature to 35 C For each reaction mix 22 ul Hybridisation buffer B 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 humidity 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 f
9. onised water Prepare the 1 x wash buffer always fresh Stability of Wash Buffer solutions 20 x Concentrate 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 1 x Working solution min 10 days at room temperature Manual Milk ID 2 0 Vers 1 0 2013 Warnings and precautions All protocol steps 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 apply 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 materia
10. ptimised for the use of HotStar Tag 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 number of PCR cycles for higher 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 electrophoreses 2 0 agarose gel Expected fragment size 115 128 bp species dependent LCD Array hybridisation and detection Detailed protocol for first time user advanced users refer to short protocol chapter 7 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
11. s 1 0 2013
12. tection by DNA incorporation hybridisation TMB precipitation Figure 1 Test principle Manual Milk ID 2 0 Vers 1 0 2013 1 2 1 3 Assay Performance The amplicons generated by the provided primer mix originate in the 16S rRNA region of the mitochondrial genomes mt DNA Due to the high copy number of the mt DNA in most tissues and the short amplicon length 125 bp the assay has to be considered as very sensitive Under standard conditions e g 35 PCR cycles for PCR amplification as little as 1 additions of one milk type into another can be detected e g 1 cow milk in 99 goat milk Since this assay has been developed for the analysis of fresh milk and products thereof increasing the PCR cycler number might become necessary if analysis of highly processed milk products is envisaged The targets amplified by the supplied PCR primer mixes are highly conserved sequences and the probes spotted on the array have been thoroughly evaluated for their species specificity for details on capture probe specificities refer to Figure 2 The probe design is based on available data base entries and sequence analysis of reference material at Chipron GmbH Due to the complexity of modern races and breeds it can not be excluded that rare genotypes escape from the analysis Array Pattern Each LCD Chip contains eight identical arrays in rectangular reaction chambers which can be addressed individually All species specific capture probes are
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