AMPLIQUALITY
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1. cia 17 12 2 1 BG DNA amplification 17 03 22 50 8033622781578 12 2 2 Direct amplification 1 amplification of Cytomegalovirus DNA 17 12 3 Visualization of the amplification products 18 12 3 1 Agarose gel electrophoresis 18 12 3 2 Sample Loading 19 12 4 INTERPRETATION OF THE RESULTS BG DNA amplification and CMV iirstamplification 20 12 5 2 Cytomegalovirus DNA amplification nested PCR 21 12 6 RESULTS INTERPRETATION second amplification of Cytomegalovirus e 22 13 TROUBLESHOOTING a 23 14 DEVIGELMES_ ee ita 25 15 DEVICE PERFORMANCES 25 15 1 SPEC 25 AM O APP 25 16 BIBLIOGRAPHIC REFERENCES eene 26 Dinamica MENT pag 2 03 22A 50 8033622781578 EN doc 1 PRODUCT INFORMATION This manual is related to the following products CMV Kit for the detection of Cytomegalovirus by DNA amplification in the UL122 region IE2 exon 4 The kit contains the reagents for DNA amplification and visualization by agarose gel electrophoresis the internal control of sample amplificability and positive control Cod Prod Pkg 03 22 25 CMV 25 test 03 22A 50 CMV 50 test CMV amplification reagents Reagents for the detection of Cytomegalovirus by DNA amplification in the UL122 region 2 exon 4 The kit contains the reagents for DNA amplification th2. ANALITICA ADVANCED BIOMEDICINE www abanalitica it USER MANUAL AMPLIQUALITY CMV ref 03 22A ref 03 22R Kit for the detection of Human Cytomegalovirus 4 0123 03 22 50 8033622781578 1 PRODUCT INFORMATION 3 2 CONTEN m 4 3 STORAGE AND STABILITY OF THE REAGENTS 5 4 PRECAUTIONS FOR USE ne pP atero cetur eaae EUER ERE esse 6 5 SAFETY RULES m as 7 5 1 General safety TUE 7 5 2 Safety rules about the kit 7 6 MATERIALS REQUIRED BUT NOT PROVIDED 9 UNE o1 9 6 2 Instruments 9 6 3 Materials A 9 7 PREPARATION OF THE 2 10 8 INTRODUGTIONI o rar e 11 9 TESTIPRINCIPLE narrazione 13 10 PRODUCT DESCRIPTION _y_r 14 11 COLLECTION MANIPULATION AND PRE TREATMENT OF THE SAMPLES C M 15 11 1 Biological liiis 15 jp 16 12 EXTRAGTIONPROTOCOL 17 121 DNA o me 17 12 2 DNA ampilification
3. 8 INTRODUCTION The understanding about biology and pathogenesis of Cytomegalovirus CMV infection have been tremendously improved first by the development of cellular culture techniques and recently by molecular biology It has been registered that the spread of the infection among population has been constantly high 40 100 but recently the number and the variety of the symptomatically evident infections cytomegalic desease have shown a great increase as consequence of the increased number of immunocompromised or immuno immature subjects immature newborn transplants malignant tumours splenectomy transfusions HIV infections old age Vancikova Z and Dvorak P 2001 In healthy subjects the CMV infection is usually asymptomatic Cytomegalovirus is a virus belonging to the Herpesviridae family it is an ubiquitous agent that infects several animal species human included and it is highly species specific CMV virus has a very slow replicative cycle and produces nuclear and cytoplasmic inclusion bodies It has a double stranded linear genome that measures about 240 Kb during the infection process in the host a specific DNA polymerase of alfa specific type is synthesized associated to a highly immunogenic protein with 3 5 exonucleasic activity is associated to it CMV genome can survive in the host cell in a latent stage with the possibility of successive reactivations otherwise it can induce a persistent infection or a ly
4. therapy is fundamental in the prevention of cytomegalic disease in particular subjects the application of more sensitive and reproducible methods such as molecular techniques was required anauitica ai pag 12 03 22A 50 8033622781578 EN doc 9 TEST PRINCIPLE PCR method Polymerase Chain Reaction has been the first method of DNA amplification described in literature Saiki RK et al 1985 It can be defined as an in vitro amplification reaction of a specific part of DNA target sequence by a thermostable DNA polymerase Three nucleic acid segments are involved in the reaction double stranded DNA template to be amplified target DNA and two single stranded oligonucleotides primers that are designed in order to anneal specifically to the template DNA The DNA polymerase begins the synthesis process at the region marked by the primers and synthesizes new double stranded DNA molecules identical to the original double stranded target DNA region by facilitating the binding and joining of the complementary nucleotides that are free in solution dNTPs After several cycles one can get millions of DNA molecules which correspond to the target sequence The sensitivity of this test makes it particularly suitable for the application in laboratory diagnostics Moreover the amplification reaction can be executed from a wide range of biological samples and since it allows to amplify very small DNA fragments the starting DNA can be als
5. bands nor for BG neither Cytomegalovirus in the tested sample but a good band for positive controls e Problems during the extraction step Be sure that the extraction kit is adequate and that you followed correctly all the instructions Consult the troubleshooting section of the extraction kit user manual Repeat the DNA extraction starting from a new sample e The amplification was inhibited Dilute the starting sample with distilled water and TE Repeat the DNA extraction from a smaller amount of clinical sample anaumica pag 23 03 22A 50 8033622781578 EN doc Use an adequate extraction system 3 Presence of aspecific products or extrabands after the visualization of the amplified products in agarose gel e The thermalcycler makes temperature changes too slowly Doa thermalcycler revision e The preparation of the amplification reaction has been executed in a long time at room temperature Accelerate the work time at room temperature Work on ice e The starting sample contained degraded DNA Repeat the extraction step using another clinical specimen Be sure that the sample had been collected and stored in appropriate Way For any further problem contact AB ANALITICA technical support e mail laboratorio abanalitica it Dinamica MENU pag 24 03 22A 50 8033622781578 EN doc 14 DEVICE LIMITS The kit can have reduced performances if e the c
6. e and water Safety data sheet MSDS of the product is available upon request Dinamica 03 22A 50 8033622781578 EN doc 6 MATERIALS REQUIRED BUT NOT PROVIDED 6 1 Reagents e Reagents for DNA extraction e Sterile DNase and RNase free water e Distilled water e Reagents for DNA visualization by agarose gel electrophoresis necessary for 03 22R kit 6 2 Instruments e Laminar flow cabinet use is recommended while adding TAQ polymerase to the amplification premix to avoid contamination it would be recommended to use another laminar flow cabinet to add the extracted DNA e Micropipettes range 0 2 2 uL 0 5 10 pL 2 20 pL e Thermalcycler e Microcentrifuge max 12 000 14 000 rpm e Balance e Magnetic heating stirrer or microwave e Chemical cabinet use is recommended in handling Ethidium Bromide e Horizontal electrophoresis chamber for agarose minigel e Power supply 50 150 V UV Transilluminator e Photo camera or image analyzer 6 3 Materials e Disposable gloves e Disposable sterile filter tips range 0 2 2 uL 0 5 10 uL 2 20 uL e Graduate cilinders 1 L for TAE dilution e Pyrex bottle or Becker for agarose gel preparation e Parafilm anaumica pag 9 m 03 22A 50 8033622781578 EN doc 7 PREPARATION OF THE REAGENTS Preparation of 1 L of 1X TAE buffer Mix 20 mL of 50X TAE with 980 mL of distilled water Dinamica MENT pag 10 03 22A 50 8033622781578 EN doc
7. e internal control of sample amplificability and the positive control Cod Prod Pkg 03 22R 25 CMV amplification reagents 25 test 03 22R 50 CMV amplification reagents 50 test anaumica pag 3 ccm 03 22A 50 8033622781578 EN doc 2 KIT CONTENT NOTE In the kits with different codes different components are included legenda X component included in the kit 0 component not included in the kit STORE AT 20 lt m COLOUR DESCRIPTION LABEL TUBE dA T o o OR LID monodose premixes for x A direct CMV amplification 20 monodose premixes for ud ux nested CMV amplification 29 ae 8 X X monodose premixes for Blue T 25 50 8 B globin gene amplification Thermostable Hot start TAQ x x oh E Sia SuperTaq Red 1x40uL 1x80uL 1x20uL pey 5 U uL STORE AT 20 lt COLOUR DESCRIPTION LABEL T o o OR LID 8 sui CMV Plasmid DNA containing part si X X positive Blue 1x30uL 1x50uL 1x 10 L of Cytomegalovirus genome confrol STORE 2 8 C lt COLOUR DESCRIPTION LABEL T o o OR LID 6X Blue Blue 4X250uL 1X450uL 1X100uL Ethidium Ethidium Bromide solution Bromide X 0 TOXIC Red 1X170uL 1X250uL 1X100uL 2 5 mg mL R 23 68 i 2 S S 36 37 45 x Mark
8. er PM Yellow 1X200uL 1X400uL 1X70uL anaumia www it pag 4 03 22A 50 8033622781578 EN doc STORE AT 15 25 lt c COLOUR DESCRIPTION LABEL SPARE 8 test db T e e OR LID X 0 Agarose Molecular Biology AGAROSE 1X20g 1X40g 1X6g grade Electrophoresis buffer X 0 TRIS Acetate EDTA pH 8 00 50X TAE 1X60mL 1X125mL 1X30mL 3 STORAGE AND STABILITY OF THE REAGENTS Each component of the kit should be stored according to the directions indicated on the label of the single boxes In particular Box P store at 20 C omall bag store at 20 C Box F store ata 2 8 C Box A store at 15 25 C When stored at the recommended temperature all test reagents are stable until their expiration date 03 22A 50 8033622781578 EN doc pag 5 4 PRECAUTIONS FOR USE eThe kit should be handled by investigator qualified through education and training in molecular biology techniques applied to diagnostics eBefore starting the kit procedure read carefully and completely the instruction manual e Keep the product away from sources of heat product out of heating sources e Do not use any part of the kit if over the expiration date e n case of any doubt about the storage conditions box integrity or method application contact ANALITICA technical support laboratorio abanalitica it In the amplificat
9. into 50 mL of 1x TAE Leave the solution on a magnetic stirring heater or in a microwave until the solution becomes clear Allow the gel to cool to hand warm and then add 10 uL of Ethidium Bromide solution 2 5 mg ml NOTICE Ethidium Bromide is a strong mutagenic agent always wear gloves and preferably work under a chemical safety cabinet during the handling of this reagent or gels containing it Place the gel into the appropriate gel casting tray with the comb placed in and allow the gel to cool at room temperature or in a fridge until the gel becomes solid When the gel is solidified remove carefully the comb pay attention not to damage the gel wells transfer the tray into the electrophoresis chamber and pour the appropriate amount of 1x TAE buffer so that it covers completely the gel about 1 2 mm over the gel surface anaumca MENO pag 18 03 22A 50 8033622781578 EN doc 12 3 2 Sample Loading Mix into a tube or directly on a parafilm layer 2 uL of 6X Blue 10 uL of amplification product or Molecular weight Marker PM marker Load the mixture in the gel wells switch on the power supply and set the voltage between 80 100 V Run the gel for about 40 min then place the gel on an UV transilluminator and analyze the results by comparing the size of the amplification products with the reference Molecular Weight Marker 6X Blue and PM Marker are included in 03 224 kit for the use of another loading buffer o
10. ion of nucleic acids the investigator has to take the following special precautions e Use filter tips e Store the biological samples the extracted DNA positive control included in the kit and all the amplification products in different places from where amplification reagents are stored eOrganise the space in different pre and post PCR units do not share consumables pipets tips tubes etc between them e Change the gloves frequently e Wash the bench surfaces with 5 sodium hypochloride e Thaw the PCR premixes at room temperature before use Add the Taq DNA polymerase and purified DNA very quickly at room temperature or in an ice bath Dinamica pag 6 03 22A 50 8033622781578 EN doc 5 SAFETY RULES 5 1 General safety rules e Wear disposable gloves to handle the reagents and the clinical samples and wash the hands at the end of work e Do not pipet with mouth eSince no known diagnostic method can assure the absence of infective agents it is a good rule to consider every clinical sample as potentially infectious and handle it as such the devices that get directly in touch with clinical samples should be considered as contaminated and disposed as such In case of accidental spilling of the samples clean up with 10 Sodium Hypochloride The materials used to clean up should be disposed in special containers for contaminated products e Clinical samples materials and contaminated produc
11. is present in low number of copies Later other publications supported these data Weinbeerg et al 2002 Cortez et al 2003 Mengelle et al 2003 The IHMF International Herpes Management Forum guidelines suggest where possibile the use of whole blood for CMV search in transplanted patients Razonable and Emery 2004 Sample collection should follow all the usual sterility precautions as routine it should be transported in sterile boxes without transport medium Blood should be treated with EDTA Other anticoagulating agents as heparin are strong inhibitors of TAQ polymerase and so they could alter the efficiency of the amplification reaction Fresh blood can be stored at 2 8 C processed within 4 hours from the collection if DNA is not shortly extracted the sample must be frozen at 20 C If the investigator prefers to start the analysis from a leukocyte pellet the Ficoll Hypaque method or the erythrocyte lysis protocol for lymphocytes isolation can be used Alternatively a buffy coat can be prepared by whole blood centrifugation at 3300 x g for 10 min at room temperature After centrifugation three different fractions can be observed the upper clear phase is the plasma the intermediate phase is the buffy coat that contains concentrated leukocytes and the lowest contains erythrocytes Dinamica MENO pag 16 03 22A 50 8033622781578 EN doc 12 12 1 DNA extraction An
12. linical sample is not suitable for this analysis not correct transport media or not correct sample storage e the DNA is not amplifiable because of the presence of inhibitors of the amplification reaction or due to an inadequate extraction system e the kit has not been stored at the suggested temperature 15 DEVICE PERFORMANCES 15 1 Specificity Primer sequence alignment in the most important databanks shows the absence of unspecific alignment and it has guaranteed the amplification of Cytomegalovirus Cross reactions with genomic DNA other pathogenic microorganism nucleic acid have not been revealed 15 2 Sensitivity With reference to other nested PCR diagnostic methods it was assumed that the sensitivity of the kit is less than 50 DNA copies 25 03 22A 50 8033622781578 EN doc 16 BIBLIOGRAPHIC REFERENCES Cortez KJ Fisher SH Fahle GA et al J Infect Dis 188 967 972 2003 Mengelle C Sandres Saune K Pasquier C et al J Clin Microbiol 41 3840 3845 2003 Mocarski ES Virology Fields BN Knipe DM Howley PM eds Philadelphia Lippincott Raven 1996 2447 2492 Razonable RR Brown RA Wilson J et al Transplantation 73 968 973 2002b Razonable RR and Emery VC Herpes 11 3 77 86 2004 Razonable RR Paya CV Smith TF J Clin Microbiol 40 746 752 2002a Saiki RK Scharf S Faloona F et al Science 230 1350 1354 1985 Vancikova Z and Dvorak P Curr Drug Targets I
13. mmune Endocr Metabol Disord 1 179 187 2001 Weinberg A Schissel D Giller R J Clin Microbiol 40 4203 4206 2002 Dinamica MENU pag 26 03 22A 50 8033622781578 EN doc ANALITICA pag 27 03 22A 50 8033622781578 EN doc 28 03 22 50 8033622781578 29 03 22A 50 8033622781578 EN doc ANALITICA AB ANALITICA srl Via Svizzera 16 35127 PADOVA ITALY e Tel 39 049 761698 Fax 39 049 8709510 e mail info abanalitica it
14. o partially degraded anaumica pag 13 03 22A 50 8033622781578 EN doc 10 PRODUCT DESCRIPTION The method proposed in this kit consists of the amplification of the viral DNA in the UL122 region The method allows also to evaluate the suitability of extracted DNA by the amplification of B globin BG gene amplification control A negative result in BG gene amplification indicates either the presence of inhibitors of the amplification reaction in the extracted DNA or that the DNA is highly degraded This method helps the operator to recognize possible false negative results for Cytomegalovirus The kit also provides with a positive control of amplification When the amplification of the positive control is successful it is guaranteed that the reaction is correct This control is not dangerous for the operator because it is plasmid DNA containing only a part of Cytomegalovirus genome The kit is in premix format all the reagents for the amplification are pre mixed and aliquoted in monodose test tubes 0 2 or 0 5 mL to which Taq polymerase and the extracted DNA will be added This premix format allows the reduction of the manipulation steps in pre amplification with considerable time saving for the operator the repeated freezing thawing of reagents that could alter the product performances is avoided and above all this format minimizes the risk of contamination so the risk to get false positive results Nevertheless it
15. oc 125 CMV nested amplification 274 DNA amplification Add to each premixed green tube 0 2 uL AB Super 1 pL First amplification product It is important to include in this step the negative and the positive controls used in the CMV direct amplification When considered useful it is possible to include also a negative control for the nested amplification by adding to the nested mix 1 uL of sterile water instead of the direct amplification product Centrifuge briefly then put the tubes into the thermalcycler programmed as below 1 cycle 95 C 5 min 95 30 35 cycles 62 C 30 sec 72 C 30 sec 1 cycle 72 C 5 min Nested amplification CMV fragment lenght 183 bp At the end of the amplification run visualize the nested amplification products by 3 agarose gel electrophoresis Weight 1 5 g of Agarose and pour it into 50 mL of 1x TAE Follow the instructions indicated at paragraph 12 3 anauitica pag 21 ccm 03 22A 50 8033622781578 EN doc 12 6 INTERPRETATION OF THE RESULTS second amplification of Cytomegalovirus DNA The controls should show the following results CONTROL RESULT INTERPRETATION Positive control Trst the second amplification are correct Negative Control NEGATIVE the first amplification Negative control Absence of contaminations in the Nested amplifica
16. oo low it could be necessary before starting DNA extraction to concentrate the sample by repeated centrifugation steps For sample resuspension sterile PBS can be used It can be useful also to remove mucus and red cells if present Collect the seminal liquid in a sterile container and store it at 4 C for some hours maximum overnight alternatively it can be frozen with addition of a suitable media for DNA extraction Urine should be collected in a sterile container stored at 2 8 C for max 24 h and then processed anaumica pag 15 03 22A 50 8033622781578 EN doc The application of molecular technique allowed better diagnosis and characterization of clinical Central Nervous System syndromes associated to CMV infection In this case the sample of choice is liquor CSF The liquor sample can be stored at room temperature or at 2 8 C if processed in a short time alternatively it is suggested to freeze the sample 11 2 Blood Since long time it was discussed about which was the blood compartment that was best indicated for CMV analysis A recent prospective study Razonable et al 2002b compared 4 blood compartments whole blood plasma PBL PBMC of 17 SOT Solid Organ Transplant and HSCT Haematopoietic Stem Cell Transplant patients and demonstrated that even if all this sample types are suitable for CMV search both for prognosis and therapy monitoring aims whole blood seems to be more sensitive when CMV
17. r of another Molecular Weight Marker follow the indications specified by the supplier DNA Molecular Weight Marker PM Marker included in 03 224 kit fragment sizes 501 489 404 353 242 190 147 110 89 67 34 26 bp NOTE 390 agarose gel the 501 489 bp bands are usually not clearly resolved and appear as an unique band the 26 and 34 bp bands are sometimes too small to be visible in a 3 agarose gel because of their low molecular weight NOTICE UV rays are dangerous for skin and above all eyes always wear gloves and safety glass or make use of the protection screen of the UV transilluminator anaumica pag 19 03 22A 50 8033622781578 EN doc 12 4 INTERPRETATION OF THE RESULTS BG amplification and CMV first amplification The controls should show the following results CONTROL RESULT INTERPRETATION Positive control POSITIVE The PCR amplification is correct Negative NEGATIVE Absence of contaminations control Then for the interpretation of the band pattern follow the table below DNA BAND RESULT INTERPRETATION BG gene band absent Sample not suitable for amplification repeat the DNA extraction BG gene band present Amplificable sample CMV band absent Proceed with the second amplification to value the true sample negativity BG gene band present Amplificable sample CMV band present positive sample for CMV aramon pag 20 03 22A 50 8033622781578 EN d
18. s always recommended to use all the proper amplification controls Dinamica MENU pag 14 03 22A 50 8033622781578 EN doc 11 COLLECTION MANIPULATION AND PRE TREATMENT OF THE SAMPLES CMV can be in free episomal or integrated form and this is determinant in the choice of the protocol to use for isolation and subsequent virus identification CMV DNA can be directly extracted from several biologic fluids like spermatic fluid maternal milk vaginal and cervical secretions saliva serum etc If starting from urine or liquor CSF a preliminary centrifugation of the sample is required to obtain a pellet enriched of cells Sometimes also the supernatant is tested If starting from peripheral blood to search CMV in leukocytes PBL or in mononuclear cells PBMC a further step for obtaining a lymphocyte pellet by FICOLL Method or red blood cells lysis is necessary CMV DNA can be extracted also from whole blood 11 1 Biological fluids In the case of cervix and vaginal swabs sample collection is performed by the use of a little swab or a little sterile brush cyto brush The collected cells are diluted in a suitable transport media 1X PBS physiological solution or other medium The sample can be stored at 2 8 C for max 48 h after then it is better to proceed with nucleic acid extraction If it is not possible to extract the sample in a short time the specimen must be stored at 20 C When sample cellularity is t
19. tic cycle during the productive inflammation the sequential expression of the viral genome sequence is time regulated by genes classified in three categories immediate early IE or alfa early E or beta and late L or gamma Mocarski ES 1996 In CMV infected patients the virus replicates in different organs as lungs liver kidney gastrointestinal tract and it is excreted through saliva breast milk spermatic fluid urine cervical and vaginal secretions tears blood and faeces The affected districts are different depending on the cause of the immunodeficiency tab 1 CMV has cytopathic effect it causes tissue damages with the formation of cytomegalic cells containing intranuclear inclusions anaumica pag 11 m 03 22A 50 8033622781578 EN doc Tab 1 CMV disease in immunosuppressed patients Razonable and Emery 2004 Symptom Newborn SOT HSCT AIDS Fever or Hepatitis Do Gastrointestinal symptoms Retinitis Pneumonia Encephalopathy Deafness Polyradiculopathy Addison disease s symptoms Transplant rejection Atherosclerosis Death SOT Solid Organ Transplant HSCT Haematopoietic stem Cell Transplant AIDS Acquired Immunodeficiency Syndrome In the past the standard methods to diagnose CMV included several techniques for virus isolation from cell cultures and for the search of pp65 antigen from peripheral blood leukocytes Razonable et al 2002a Since the
20. tion second amplification Then for the interpretation of the band pattern follow the table below DNA BAND RESULT INTERPRETATION CMV band in the first and spear Negative sample for second amplification Cytomegalovirus CMV band in the second Positive sample for amplification present Cytomegalovirus Fig 1 396 agarose gel products electrophoresis of the amplification 1 DNA Molecular Weight Marker MW 2 Positive control for CMV DNA 3 CMV positive sample 4 Negative control pag 22 03 22A 50 8033622781578 EN doc 13 TROUBLESHOOTING 1 Neither amplification products nor positive control DNA band e TAQ polymerase was not correctly added to the premix Use pipets and tips of suitable volume range pipet range 0 2 2 uL Check visually that TAQ polymerase diffuses in the premix this is easy because the enzyme is dissolved in glycerol that has a higher density Alternatively check visually the drop of TAQ polymerase put on the tube wall then centrifuge briefly e The thermalcycler was not correctly programmed Check the conformity of the thermalcycler program and the temperature profile in the instruction manual then repeat the amplification with the correct program e The kit doesn t work properly Store the premix TAQ polymerase and positive control at 20 C Avoid repeated freezing thawing of the premix and the reagents 2 No amplification
21. ts should be disposed after decontamination by immersion in a solution of 596 Sodium Hypochloride 1 volume of 596 Sodium Hypochloride solution every 10 volumes of contaminated fluid for 30 minutes OR autoclaving at 121 C at least for 2 hours NOTE do not autoclave solutions containing Sodium Hypochloride 5 2 Safety rules about the kit The risks for the use of this kit are related to the single components Dangerous components ETHIDIUM BROMIDE included in the 03 224 kit 3 8 diamino 1 ethyl 6 phenylphenantridiumbromide 296 Description of risk Toxic 2 anaumca pag 7 03 22A 50 8033622781578 EN doc RISK SENTENCES AND S SENTENCES ETHIDIUM BROMIDE R23andR68 Toxic for inhalation Risk of irreversible effects S 36 37 45 Wear laboratory coat and disposable gloves In case of accident or discomfort seek for medical assistance and show the package label R and S sentences refer to the concentrated product as provided in the kit In particular for Ethidium Bromide until the dilution in the agarose gel In manipulating concentrated Ethidium Bromide use a chemical dispensing fume cabinet Always wear disposable gloves and laboratory coat in manipulating the diluted Ethidium solution as well The product can not be disposed with the common waste It must not reach the drainer system For the disposal follow the local law In case of accidental spilling of Ethidium Bromide clean with Sodium hypochlorid
22. y extraction method can be used provided that it extracts pure and integral DNA It is necessary to remember that the volumes of the extracted DNA indicated in paragraph 12 2 for BG and CMV amplification are referred to the extracts obtained with ANALTICA methods Using an alternative extraction method if the volume of the DNA solution to amplify is less than 10 uL it will be necessary to adjust the volume of the amplification mix by adding water 12 2 amplification 12 2 1 BG DNA amplification For each sample add to each premixed tube blue tube 0 2 uL AB Super 10 uL extracted DNA 12 2 2 Direct amplification 1 amplification of DNA For each sample add to each premixed red tube 0 2 uL AB Super 10 uL extracted DNA It is important to include in each experiment a negative control add to the mix distilled water instead of DNA and a positive control 10 uL of the positive control included in the kit 17 ccm 03 22A 50 8033622781578 EN doc Centrifuge briefly put the tubes BG and CMV into the thermalcycler programmed as below 1 cycle 95 C 5 min 95 60 sec 40 cycles 56 60 sec 72 C 60 sec 1 cycle 72 C 5 min Amplification products lenght CMV 472 bp BG 268 bp 12 3 Visualization of the amplification products 12 3 1 Agarose gel electrophoresis Prepare a 3 agarose gel Weight 1 5 g of Agarose and pour it
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