Escherichia coli O157:H7 Kayan Gane Acid Nucleic Acid (Tsarin Binciken Fluorescent PCR)
Bayanin Kit:
Cat. No.FP102
Ana amfani dashi don ganowa da sauri da kuma nunawa E. coli O157: H7 a cikin abinci, abinci, samfurori na ruwa da samfurori na muhalli.
Bayani
Ana amfani dashi don ganowa da sauri da kuma nunawa E. coli O157: H7 a cikin abinci, abinci, samfurori na ruwa da samfurori na muhalli.
[Tsarin gwaji]Bisa ga ka'idar fasahar PCR mai kyalli, an tsara ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun bayanai da bincike na Taqman don takamaiman nau'in Escherichia coli O157: H7, kuma an gano shi ta kayan aikin PCR mai kyalli, don gane gano Escherichia coli O157: H7 Ingantacciyar gano DNA.
Abubuwan da ke cikin Kit
Lura: Ba a haɗa binciken tashar ROX ba.
| Cmasu kai hari | Ƙayyadaddun bayanai | Quantity |
| Buffer A | Tube | 1 |
| Buffer B | Tube | 1 |
| Kyakkyawan iko | Tube | 1 |
| Sarrafa mara kyau | Tube | 1 |
Amfanin da ake tsammani
Ana amfani dashi don saurin ganowa da nunawa E. coli O157: H7 a cikin abinci, abinci, samfuran ruwa da samfuran muhalli.
Yanayin Ajiya Da Ranar Karewa
Ajiye a -20 ℃ a cikin duhu kuma kauce wa daskarewa da narke maimaituwa.
Lokacin tabbatarwa shine 12watanni, kuma ana nuna ranar samarwa akan marufi na waje.
Instruments Da Abubuwan Amfani
Kayan aikin PCR mai walƙiya, bindigar pipette da madaidaitan tukwici, girgizar vortex, ƙaramin centrifuge.
Amfani
1. Samfuran Gudanarwa
1.1 Samfurin Nau'in: Wannan kit ɗin ya dace da abinci, abinci, samfuran ruwa da sauran samfuran da ake zargin Escherichia coli O157: H7 ya gurɓata.Don samfuran naman da aka sarrafa mai zurfi, abubuwan sha da sauran abubuwan da ke ɗauke da pigments, suna buƙatar kurkure su don guje wa tasirin tarin siginar walƙiya.
1.2 Samfurin sarrafawa: Koma zuwa "GB 4789.10-2016 Tsaron Abinci na Ƙasa Standard Food Microbiological Examination of Escherichia coli O157: H7 Testing" don samfurin shirye-shiryen, al'adar wadata da warewar Escherichia coli O157: H7.
- Nucleic acid hakar
Ɗauki 20 ml na maganin haɓakawa a cikin bututun centrifuge na 1.5 ml, ƙara 200 μL na microbial lysate (ana buƙatar ƙarin kayan aiki), vortex na 30 seconds, centrifuge a taƙaice, kuma ajiye shi.
Bayani: Cire acid nucleic daga lysate ya kamata a kammala a cikin minti 10, kuma ba za a iya adana shi na dogon lokaci ba.
3. Nucleic Acid Amplification
3.1 Kunna kayan aikin PCR mai kyalli don amfani.
Buffer A da Buffer B daga kayan kit, narke su sosai, kuma a saka su a taƙaice.Ƙara 18 μL Buffer A da 2 μL Buffer B zuwa kowane bututu mai amsawa na PCR.Sa'an nan kuma ƙara 5 ml kowanne daga cikin mummunan iko, fitar da nucleic acid, da kuma tabbatacce iko a cikin PCR dauki bututu, hula da tubes, da centrifuge a takaice.
3.3 Canja wurin bututun amsawar PCR zuwa injin PCR mai kyalli, kuma amfani da waɗannan hanyoyin don yin gwaje-gwajen haɓakawa: zaɓi 25 ml don tsarin amsawa, tattara siginar kyalli a 60 ° C ga kowane sake zagayowar, kuma zaɓi FAM don tashar ganowa.
| Mataki | Shirin | Yawan hawan keke |
| 1 | 37 ℃ 5 min | 1 |
| 2 | 9 5 ℃ 3 min | 1 |
| 3 | 95°C 15s | 40 |
| 60 ℃ 30s (tattara haske) |
Jagora don nazarin matsalolin
The following is an analysis of the problems that might be encountered in the extraction of viral RNA. We wish it would be helpful to your experiment. In addition, for other experimental or technical problems other than operating instructions and problem analysis, we have dedicated technical support to help you. Contact us if you need at : 028-83361257or E-mail：Tech@foregene.com。
Ba za a iya fitar da RNA ba ko yawan amfanin acid nucleic ya yi ƙasa
Yawancin lokaci akwai dalilai da yawa waɗanda ke shafar ingancin farfadowa, kamar: samfurin abun ciki na RNA, hanyar aiki, ƙarar haɓakawa, da sauransu.
Binciken dalilai na gama gari:
1.Ice wanka ko ƙananan zafin jiki (4 ° C) centrifugation yayin aiki.
Shawarwari: Yanayin zafin jiki (15-25 ° C) aiki, ba wankan kankara da ƙananan zafin jiki ba.
2. Samfurin da ba daidai ba ko ajiyar samfurin na dogon lokaci.
Shawara: Ajiye samfurori a -80 ° C ko daskare a cikin ruwa nitrogen, kuma kauce wa daskare-narke maimaita amfani;gwada amfani da sabbin samfuran da aka tattara don hakar RNA.
3. Rashin isasshen samfurin lysis
Shawarwari: Da fatan za a tabbatar da cewa samfurin da maganin aiki (Linear Acrylamide) an haɗa su sosai kuma an sanya su na minti 10 a zazzabi na ɗaki (15-25 ° C)
4.An ƙara eluent ba daidai ba
Shawarwari: Tabbatar cewa an ƙara ddH2O-Free RNase zuwa tsakiyar membrane na ginshiƙin tsarkakewa.
5.Ba daidai ba girma na anhydrous ethanol a cikin Buffer viRW2
Shawara: Da fatan za a bi umarnin, ƙara madaidaicin ƙarar ethanol mai anhydrous zuwa Buffer viRW2 kuma a haɗa su da kyau kafin amfani da kit ɗin.
6.Yin amfani da samfur mara kyau.
Shawara: 200µl na samfurin a kowace 500μl na Buffer viRL.Ƙarfin samfurin da ya wuce kima zai haifar da raguwar adadin hakar RNA.
7.Ba daidai ba ƙarar ƙira ko rashin cikawa.
Shawarwari: Ƙaƙwalwar haɓakar ginshiƙin tsarkakewa shine 30-50μl;idan tasirin elution bai gamsar ba, ana ba da shawarar ƙara ddH mai zafi-Free RNase.2O kuma ƙara lokacin sanyawa a zafin jiki, kamar minti 5-10
8.Purification shafi yana da ragowar ethanol bayan kurkura a cikin Buffer viRW2.
Shawara: Idan har yanzu ethanol ya kasance bayan kurkura a cikin Buffer viRW2 da fanko-tube centrifugation na 2min, za'a iya barin ginshiƙin tsarkakewa a cikin zafin jiki na 5min bayan centrifugation na bututu don cikakken cire sauran ethanol.
Lalacewar ƙwayoyin RNA masu tsafta
Ingancin RNA da aka tsarkake yana da alaƙa da abubuwa kamar ajiyar samfur, gurɓataccen RNase, da aiki.
Binciken dalilai na gama gari:
1.Ba a adana samfurori da aka tattara a lokaci ba.
Shawara: Idan ba a yi amfani da samfurin a cikin lokaci bayan tattarawa ba, da fatan za a adana shi a -80 ℃ ko nitrogen ruwa nan da nan.Don hakar kwayoyin RNA, gwada amfani da samfurori da aka tattara a duk lokacin da zai yiwu.
2. Samfuran da aka tattara suna daskarewa kuma suna narke akai-akai.
Shawara: A guji maimaita daskarewa da narke (ba fiye da sau ɗaya ba) yayin tattara samfuri da adanawa, in ba haka ba yawan adadin acid nucleic zai ragu.
An gabatar da 3.RNase a cikin dakin aiki ko kuma ba a sa safar hannu, abin rufe fuska, da sauransu.
Shawara: An fi yin hakar gwajin kwayoyin RNA a cikin wani dakin aikin RNA daban, kuma ana tsaftace teburin gwajin kafin gwajin.Saka safofin hannu da abin rufe fuska yayin gwajin don guje wa lalatawar RNA ta hanyar gabatarwar RNase.
4.The reagent yana gurbata ta RNase yayin amfani.
Shawara: Sauya da sabon Katin Keɓewar RNA na ƙwayar cuta don gwaje-gwaje masu alaƙa.
5.Cibiyar RNase na bututun centrifuge, tukwici na pipette, da sauransu. Shawara: Tabbatar cewa bututun centrifuge, tukwici na pipette, da pipettes duk ba su da RNase.
Kwayoyin RNA da aka tsarkake sun shafi gwaje-gwaje na ƙasa
Kwayoyin RNA da aka tsarkake ta hanyar ginshiƙin tsarkakewa za su yi tasiri ga gwaje-gwajen da ke ƙasa idan akwai ions gishiri ko sunadaran da yawa, kamar: fassarar juzu'i, Northern Blot, da sauransu.
1.Akwai sauran ions gishiri a cikin kwayoyin RNA da aka eluted.
Shawarwari: Tabbatar cewa an ƙara madaidaicin ƙarar ethanol anhydrous zuwa Buffer viRW2, kuma ku wanke ginshiƙin tsarkakewa sau biyu bisa ga madaidaicin saurin centrifugation akan umarnin aiki;Idan har yanzu akwai sauran ions gishiri, zaku iya ƙara Buffer viRW2 zuwa ginshiƙin tsarkakewa, kuma ku bar shi a cikin dakin zafin jiki na 5min.Sa'an nan kuma yi centrifugation don cire gurɓatar ions gishiri zuwa mafi girma
2.Akwai sauran ethanol a cikin kwayoyin RNA da aka eluted
Shawara: da zarar an tabbatar da cewa Buffer viRW2 ya wanke ginshiƙan tsarkakewa, aiwatar da centrifugation na fanko bisa ga saurin centrifugal akan umarnin aiki.Idan har yanzu akwai sauran ethanol, ana iya barin shi na mintuna 5 a zafin jiki na ɗaki bayan centrifugation na bututu don cire sauran ethanol zuwa mafi girma.
Jagoran Jagora:
Littafin Umarnin Keɓewa na RNA Viral
