Escherichia coli O157: H7 Nucleic Acid Detection Kit (Njira Yofufuza ya Fluorescent ya PCR)
Kufotokozera Kwa Zida:
Mphaka No.Chithunzi cha FP102
Amagwiritsidwa ntchito pozindikira msanga ndi kuwunika kwa E. coli O157: H7 muzakudya, chakudya, zitsanzo za madzi ndi zitsanzo zachilengedwe.
Kufotokozera
Amagwiritsidwa ntchito pozindikira msanga ndi kuwunika kwa E. coli O157: H7 mu chakudya, chakudya, zitsanzo za madzi ndi zitsanzo zachilengedwe.
[Mfundo yoyesera]Malinga ndi mfundo yaukadaulo wa PCR wa fulorosenti, zoyambira zenizeni ndi ma probe a Taqman amapangidwira jini yeniyeni ya Escherichia coli O157: H7, ndikuzindikiridwa ndi chida cha fulorosenti ya PCR, kuti azindikire kuzindikira kwa Escherichia coli O157: H7 Kuzindikira koyenera kwa DNA.
Zamkatimu Zamkati
Chidziwitso: kafukufuku wamakanema a ROX sanaphatikizidwe.
| Cotsutsa | Kufotokozera | Qunity |
| Buffer A | Chubu | 1 |
| Buffer B | Chubu | 1 |
| Kulamulira kwabwino | Chubu | 1 |
| Kuwongolera kolakwika | Chubu | 1 |
Kugwiritsa Ntchito Kuyembekezeredwa
Amagwiritsidwa ntchito kuzindikira mwamsanga ndi kuunika kwa E. coli O157: H7 mu chakudya, chakudya, zitsanzo za madzi ndi zitsanzo zachilengedwe.
Zosungirako Ndi Tsiku Lotha Ntchito
Sungani pa -20 ℃ mumdima ndipo pewani kuzizira mobwerezabwereza ndi kusungunuka.
Nthawi yovomerezeka ndi 12miyezi, ndipo tsiku lopanga likuwonetsedwa pamatumba akunja.
Zida Ndi Zogwiritsidwa Ntchito
Chida cha Fluorescent quantitative PCR, mfuti ya pipette ndi malangizo ofananira, vortex shaker, mini centrifuge.
Kugwiritsa ntchito
1. Zitsanzo Processing
1.1 Mtundu wachitsanzo: Chida ichi ndi choyenera chakudya, chakudya, zitsanzo za madzi ndi zitsanzo zina zomwe zikuganiziridwa kuti zakhudzidwa ndi Escherichia coli O157: H7.Pazakudya zozama kwambiri za nyama, zakumwa ndi zinthu zina zokhala ndi inki, ziyenera kutsukidwa kuti zisasokoneze chizindikiro cha fluorescence.
1.2 Kukonza Zitsanzo: Onani "GB 4789.10-2016 Food Safety National Standard Food Microbiological Examination of Escherichia coli O157: H7 Test" pokonzekera zitsanzo, chikhalidwe cholemeretsa komanso kudzipatula kwa Escherichia coli O157: H7.
- Nkuchotsa ucleic acid
Tengani 20 ml ya njira yowonjezeretsa mu chubu cha 1.5 mL centrifuge, onjezerani 200 μL wa microbial lysate (zowonjezera zida zofunika), vortex kwa masekondi 30, centrifuge mwachidule, ndikuyika pambali.
Ndemanga: Kutulutsa kwa nucleic acid kuchokera ku lysate kuyenera kumalizidwa mkati mwa mphindi 10, ndipo sikungasungidwe kwa nthawi yayitali.
3. Nucleic Acid Amplification
3.1 Yatsani chida cha fulorosenti cha PCR kuti mugwiritse ntchito.
Buffer A ndi Buffer B kuchokera ku zida, zisungunuleni bwino, ndi centrifuge mwachidule.Onjezani 18 μL Buffer A ndi 2 μL Buffer B ku chubu chilichonse cha PCR.Kenako onjezani 5 mL chilichonse chowongolera choyipa, chochotsa nucleic acid, ndikuwongolera bwino mu machubu a PCR, kapuni machubu, ndi centrifuge mwachidule.
3.3 Tumizani chubu cha PCR pamakina a fulorosenti a PCR, ndipo gwiritsani ntchito njira zotsatirazi kuti muyese kuyesa kukulitsa: sankhani 25 mL kuti mugwiritse ntchito, sonkhanitsani zizindikiro za fluorescence pa 60 ° C pamtundu uliwonse, ndikusankha FAM ya njira yodziwira.
| Khwerero | Pulogalamu | Chiwerengero cha zozungulira |
| 1 | 37 ℃ 5 min | 1 |
| 2 | 9 5 ℃ 3 min | 1 |
| 3 | 95 ° C 15s | 4 0 |
| 60 ℃ 30s (sonkhanitsani fulorosenti) |
Malangizo owunikira mavuto
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。
Palibe RNA yomwe ingatulutsidwe kapena zokolola za nucleic acid ndizochepa
Nthawi zambiri pamakhala zinthu zambiri zomwe zimakhudza kuchira bwino, monga: zitsanzo za RNA, njira yogwirira ntchito, voliyumu, etc..
Kusanthula kwazomwe zimayambitsa:
1.Ice kusamba kapena otsika kutentha (4 ° C) centrifugation pa ntchito.
Malangizo: Kutentha kwachipinda (15-25 ° C), osasamba madzi oundana komanso kutentha kwa centrifuge.
2. Kusungidwa kwachitsanzo kosayenera kapena kusungirako zitsanzo kwa nthawi yayitali.
Malangizo: Sungani zitsanzo pa -80 ° C kapena kuzizira mu nayitrogeni wamadzimadzi, ndipo pewani kugwiritsa ntchito kuzizira kobwerezabwereza;yesani kugwiritsa ntchito zitsanzo zomwe zasonkhanitsidwa mwatsopano kuti muchotse RNA.
3.Kusakwanira kwachitsanzo lysis
Malangizo: Chonde onetsetsani kuti zitsanzo ndi njira yogwirira ntchito (Linear Acrylamide) yasakanizidwa bwino ndikuyimitsidwa kwa mphindi 10 kutentha (15-25 ° C)
4.Chidziwitso chinawonjezedwa molakwika
Malangizo: Onetsetsani kuti RNase-Free ddH2O yawonjezedwa pakati pa nembanemba ya gawo loyeretsa.
5.Voliyumu yolakwika ya ethanol ya anhydrous mu Buffer viRW2
Yesani: Chonde tsatirani malangizo, onjezani voliyumu yolondola ya ethanol ya anhydrous ku Buffer viRW2 ndikusakaniza bwino musanagwiritse ntchito zida.
6.Kugwiritsira ntchito chitsanzo molakwika.
Malingaliro: 200µl ya zitsanzo pa 500μl ya Buffer viRL.Kuchuluka kwachitsanzo kumabweretsa kuchepa kwa RNA m'zigawo.
7. Voliyumu yolakwika kapena kusakwanira.
Lingaliro: Voliyumu yosawoneka bwino ya gawo loyeretsera ndi 30-50μl;ngati mphamvu ya elution siyikukhutiritsa, tikulimbikitsidwa kuwonjezera RNase-Free ddH yotenthetserapo.2O ndikuwonjezera nthawi yoyika kutentha, monga 5-10min
8.Kuyeretsa kumakhala ndi zotsalira za ethanol mutatsuka mu Buffer viRW2.
Lingaliro: Ngati Mowa utsalirabe mutatha kutsuka mu Buffer viRW2 ndi centrifugation yopanda kanthu-chubu kwa 2min, ndime yoyeretsera imatha kusiyidwa kutentha kwa mphindi 5 pambuyo pa chubu chopanda kanthu kuti muchotse ethanol yotsalira.
Kuwonongeka kwa mamolekyu oyeretsedwa a RNA
Ubwino wa RNA yoyeretsedwa umagwirizana ndi zinthu monga kusungirako zitsanzo, kuipitsidwa kwa RNase, ndi ntchito.
Kusanthula kwazomwe zimayambitsa:
1.Zitsanzo zosonkhanitsidwa sizinasungidwe mu nthawi.
Yesani: Ngati chitsanzocho sichigwiritsidwa ntchito pakapita nthawi, chonde sungani pa -80 ℃ kapena nayitrogeni wamadzimadzi nthawi yomweyo.Pochotsa mamolekyu a RNA, yesani kugwiritsa ntchito zitsanzo zomwe zasonkhanitsidwa mwatsopano ngati kuli kotheka.
2.Zitsanzo zosonkhanitsa zinali kuzizira ndi kusungunuka mobwerezabwereza.
Yesani: Pewani kuzizira kobwerezabwereza ndi kusungunuka (osapitirira kamodzi) panthawi yosonkhanitsa ndi kusunga, apo ayi zokolola za nucleic acid zidzachepa.
3.RNase idayambitsidwa m'chipinda chopangira opaleshoni kapena palibe magolovesi otaya, masks, ndi zina zambiri.
Lingaliro: Kutulutsa kwa kuyesa kwa mamolekyu a RNA kumachitidwa bwino m'chipinda chapadera cha RNA, ndipo tebulo loyesera limatsukidwa kuyesako kusanachitike.Valani magolovesi ndi masks otayika panthawi yoyesera kuti mupewe kuwonongeka kwa RNA komwe kumachitika chifukwa cha kuyambika kwa RNase.
4.The reagent imakhudzidwa ndi RNase panthawi yogwiritsira ntchito.
Yesani: Bwezerani ndi Viral RNA Isolation Kit yatsopano pazoyeserera zofananira.
5.Kuwonongeka kwa RNase kwa machubu a centrifuge, nsonga za pipette, ndi zina zotero.
Mamolekyu oyeretsedwa a RNA adakhudza zoyeserera zakutsika
Mamolekyu a RNA oyeretsedwa ndi gawo loyeretsera adzakhudza kuyesa kumtunda ngati pali ma ion amchere ambiri kapena mapuloteni, monga: reverse transcript, Northern Blot, etc..
1.Pali ma ion amchere otsala mu ma molekyulu a RNA opangidwa.
Malangizo: Onetsetsani kuti voliyumu yolondola ya ethanol ya anhydrous yawonjezedwa ku Buffer viRW2, ndikutsuka ndime yoyeretsa kawiri molingana ndi liwiro lolondola la centrifugation pamalangizo opangira; Ngati pali ma ion amchere otsala, mutha kuwonjezera Buffer viRW2 pagawo loyeretsera, ndikuyisiya kutentha kwa 5min.Ndiye kuchita centrifugation kuchotsa mchere ayoni kuipitsidwa kwambiri
2.Pali ethanol yotsala mu mamolekyu a RNA opangidwa
Lingaliro: mutatsimikizira kuti mizati yoyeretsera yatsukidwa ndi Buffer viRW2, chitani chopanda kanthu-chubu centrifugation malinga ndi liwiro la centrifugal pa malangizo ogwiritsira ntchito.Ngati pali Mowa otsala, akhoza kusiyidwa kwa mphindi 5 kutentha firiji pambuyo chopanda kanthu chubu centrifugation kuchotsa Mowa otsala kumlingo waukulu.
Mabuku a Malangizo:
Viral RNA Isolation Kit Instruction Manual
