Escherichia coli O157: H7 Nucleic Acid Detection Kit (PCR Fluorescent Probe Method)
Kit Description:
Cat. No.PAB 102
Nws yog siv rau kev tshawb nrhiav sai thiab tshuaj ntsuam xyuas E. coli O157:H7 hauv cov zaub mov, pub, cov qauv dej thiab cov qauv ib puag ncig.
Cov lus piav qhia
Nws yog siv rau kev tshawb nrhiav sai thiab tshuaj ntsuam xyuas E. coli O157: H7 hauv cov zaub mov, pub, cov qauv dej thiab cov qauv ib puag ncig.
[Tsim qauv]Raws li lub hauv paus ntsiab lus ntawm fluorescent PCR thev naus laus zis, cov primers tshwj xeeb thiab Taqman sojntsuam yog tsim los rau cov noob tshwj xeeb ntawm Escherichia coli O157: H7, thiab kuaj pom los ntawm cov cuab yeej fluorescent PCR, thiaj li paub txog kev tshawb pom ntawm Escherichia coli O157: H7 Kev kuaj pom zoo ntawm DNA.
Cov khoom siv
Nco tseg: ROX channel sojntsuam tsis suav nrog.
| Ccov omponents | Specification | Qmuaj nuj nqis |
| Buffer A | Tube | 1 |
| Tsis B | Tube | 1 |
| Kev tswj zoo | Tube | 1 |
| Kev tswj tsis zoo | Tube | 1 |
Xav siv
Nws yog siv rau Kev tshawb nrhiav sai thiab tshuaj ntsuam xyuas ntawm E. coli O157: H7 hauv cov khoom noj, pub, cov qauv dej thiab cov qauv ib puag ncig.
Cia Lub Sijhawm Thiab Hnub Kawg
Khaws ntawm -20 ℃ nyob rau hauv qhov tsaus ntuj thiab tsis txhob rov qab khov thiab thawing.
Lub sijhawm siv tau yog 12lub hlis, thiab hnub tsim khoom yog qhia nyob rau hauv lub ntim txheej.
Instruments Thiab Consumables
Fluorescent quantitative PCR ntsuas, phom pipette thiab cov lus qhia sib txuam, vortex shaker, mini centrifuge.
Kev siv
1. Qauv ua
1.1 Hom qauv: Cov khoom siv no tsim nyog rau cov khoom noj, pub, cov qauv dej thiab lwm yam qauv uas xav tias yuav kis tau los ntawm Escherichia coli O157:H7.Rau cov khoom siv nqaij sib sib zog nqus, dej haus thiab lwm yam khoom uas muaj cov xim, lawv yuav tsum tau muab yaug kom tsis txhob cuam tshuam cov teeb liab fluorescence.
1.2 Qauv ua: Xa mus rau "GB 4789.10-2016 Food Safety National Standard Food Microbiological Examination of Escherichia coli O157: H7 Test" rau cov qauv kev npaj, enrichment kab lis kev cai thiab cais ntawm Escherichia coli O157: H7.
- Nextraction ntawm ukleic acid
Noj 20 mL ntawm cov tshuaj enrichment hauv 1.5 mL centrifuge raj, ntxiv 200 μL ntawm microbial lysate (cov khoom siv ntxiv yog xav tau), vortex rau 30 vib nas this, centrifuge luv luv, thiab tso tseg.
Lus Cim: Kev rho tawm ntawm nucleic acid los ntawm lysate yuav tsum ua kom tiav hauv 10 feeb, thiab tsis tuaj yeem khaws cia ntev.
3. Nucleic Acid Amplification
3.1 Qhib cov khoom siv hluav taws xob fluorescent quantitative PCR rau kev siv.
Buffer A thiab Buffer B los ntawm cov khoom siv, yaj lawv kom huv si, thiab centrifuge luv luv.Ntxiv 18 μL Buffer A thiab 2 μL Buffer B rau txhua lub raj PCR.Tom qab ntawd ntxiv 5 mL txhua qhov kev tswj tsis zoo, tshem tawm cov nucleic acid, thiab kev tswj zoo rau hauv PCR cov tshuaj tiv thaiv kab mob, hau cov hlab, thiab centrifuge luv luv.
3.3 Hloov lub PCR cov tshuaj tiv thaiv raj mus rau lub tshuab fluorescent PCR, thiab siv cov txheej txheem hauv qab no los ua cov kev sim ua kom muaj zog: xaiv 25 mL rau cov tshuaj tiv thaiv kab mob, sau cov teeb liab fluorescence ntawm 60 ° C rau txhua lub voj voog, thiab xaiv FAM rau kev kuaj pom channel.
| Kauj ruam | Program | Tus lej ntawm lub voj voog |
| 1 | 37 ℃ 5 feeb | 1 |
| 2 | 95 ℃ 3 feeb | 1 |
| 3 | 95°C 15s | 4 0 |
| 60 ℃ 30s (sau fluorescence) |
Cov lus qhia rau kev txheeb xyuas qhov teeb meem
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。
Tsis muaj RNA tuaj yeem muab rho tawm lossis cov txiaj ntsig ntawm nucleic acid tsawg
Feem ntau muaj ntau yam uas cuam tshuam rau kev rov ua haujlwm zoo, xws li: qauv RNA cov ntsiab lus, txoj kev ua haujlwm, qhov ntim elution, thiab lwm yam.
Analysis ntawm common ua:
1.Ice da dej los yog qhov kub thiab txias (4 ° C) centrifugation thaum ua haujlwm.
Tswv yim: Chav ntsuas kub (15-25 ° C) ua haujlwm, tsis txhob da dej da dej thiab kub centrifuge.
2. Cov qauv khaws cia tsis raug lossis cov qauv khaws cia ntev dhau.
Tswv yim: Khaws cov qauv ntawm -80 ° C los yog khov hauv cov kua nitrogen, thiab tsis txhob rov siv dua khov-thaw;sim siv cov qauv sau tshiab rau RNA rho tawm.
3.Cov qauv lysis tsis txaus
Cov lus pom zoo: Thov xyuas kom meej tias cov qauv thiab cov kev daws teeb meem ua haujlwm (Linear Acrylamide) tau muab sib xyaw kom zoo thiab incubated rau 10 min ntawm chav tsev kub (15-25 ° C)
4.Qhov eluent tau ntxiv tsis raug
Kev pom zoo: Nco ntsoov tias RNase-Free ddH2O ntxiv rau hauv nruab nrab ntawm daim nyias nyias ntawm kab purification
5.Improper ntim ntawm anhydrous ethanol hauv Buffer viRW2
Tswv yim: Thov ua raws li cov lus qhia, ntxiv qhov tseeb ntim ntawm anhydrous ethanol rau Buffer viRW2 thiab sib tov zoo ua ntej siv cov khoom siv.
6. Cov qauv siv tsis raug.
Tswv yim: 200µl ntawm cov qauv ib 500μl ntawm Buffer viRL.Cov qauv ntim ntau dhau yuav ua rau txo RNA rho tawm tus nqi.
7.Improper elution ntim lossis tsis tiav elution.
Tswv yim: Lub ntim eluent ntawm purification kem yog 30-50μl;Yog tias cov nyhuv elution tsis txaus siab, nws raug nquahu kom ntxiv pre-rhuab RNase-Free ddH2O thiab ncua sij hawm tso rau hauv chav tsev kub, xws li 5-10min
8.Purification kem muaj ethanol residue tom qab yaug hauv Buffer viRW2.
Cov lus pom zoo: Yog tias ethanol tseem tseem nyob tom qab yaug tawm hauv chav nyob hauv chav tsev rau 5min, colation-raj centrifugation rau tag nrho cov ethanol.
Lub degradation ntawm purified RNA molecules
Qhov zoo ntawm RNA purified muaj feem xyuam nrog cov khoom xws li cov qauv cia, RNase paug, thiab kev ua haujlwm.
Analysis ntawm common ua:
1.Cov qauv sau tseg tsis tau txais kev cawmdim raws sijhawm.
Cov lus qhia: Yog tias tus qauv tsis siv sijhawm tom qab sau, thov khaws cia ntawm -80 ℃ lossis kua nitrogen tam sim.Rau kev rho tawm ntawm RNA molecules, sim siv cov qauv sau tshiab thaum twg los tau.
2.Cov qauv sau tau khov thiab thawing ntau zaus.
Tswv yim: Tsis txhob rov ua kom khov thiab thawing (tsis pub ntau tshaj ib zaug) thaum lub sij hawm coj mus kuaj thiab cia, txwv tsis pub cov tawm los ntawm nucleic acid yuav txo.
3.RNase tau qhia hauv chav ua haujlwm lossis tsis muaj hnab looj tes pov tseg, qhov ncauj qhov ntswg, thiab lwm yam.
Tswv yim: Kev rho tawm ntawm RNA molecules kev sim ua tau zoo tshaj plaws hauv chav ua haujlwm RNA cais, thiab cov lus sim raug ntxuav ua ntej kev sim.Hnav hnab looj tes thiab lub qhov ncauj qhov ntswg pov tseg thaum lub sij hawm sim kom tsis txhob RNA degradation los ntawm RNase qhia.
4.Cov reagent muaj paug los ntawm RNase thaum siv.
Tswv yim: Hloov tshiab Viral RNA Isolation Kit rau lwm yam kev sim.
5.The RNase paug ntawm cov raj centrifuge, cov lus qhia pipette, thiab lwm yam. Cov lus qhia: Xyuas kom tseeb tias cov raj centrifuge, cov lus qhia pipette, thiab pipettes yog tag nrho RNase-Free.
Cov purified RNA molecules cuam tshuam cov kev sim hauv qab
RNA molecules purified los ntawm kab purification yuav cuam tshuam cov kev sim downstream yog tias muaj ntsev ions ntau dhau los yog cov proteins, xws li: thim rov qab, Northern Blot, thiab lwm yam.
1.Muaj cov ntsev ions ntxiv hauv cov eluted RNA molecules.
Kev pom zoo: Xyuas kom tseeb tias qhov ntim ntawm cov dej tsis muaj dej ethanol tau muab ntxiv rau Buffer viRW2, thiab ntxuav cov purification kem ob zaug raws li qhov tseeb centrifugation ceev ntawm cov lus qhia ua haujlwm; Yog tias tseem muaj ntsev ions ntxiv, koj tuaj yeem ntxiv Buffer viRW2 rau hauv kem purification, thiab tawm hauv chav sov li 5 feeb.Tom qab ntawd ua centrifugation kom tshem tawm cov ntsev ions paug rau qhov loj tshaj plaws
2.Muaj ethanol nyob rau hauv eluted RNA molecules
Cov lus qhia: ib zaug lees paub tias cov kab huv huv tau muab yaug los ntawm Buffer viRW2, ua cov raj khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob.Yog tias tseem muaj ethanol tseem tshuav, nws tuaj yeem tso tseg rau 5 feeb ntawm chav tsev kub tom qab lub raj khoob khoob khoob kom tshem tawm cov ethanol seem mus rau qhov loj tshaj plaws.
Phau ntawv qhia:
Viral RNA Isolation Kit Instruction Manual
