I-Escherichia Coli O157: I-H7 Nucleic Acid Detection Kit (I-PCR Fluorescent Probe Method/Lyophilization)
Inkcazelo yeKit:
Ikati.No.FP103
Isetyenziselwa ukukhangela ngokukhawuleza kunye nokuhlolwa kwe-E. coli O157: H7 ekutyeni, ukutya, iisampulu zamanzi kunye neesampuli zokusingqongileyo.
Iinkcazelo
Isetyenziselwaukufunyanwa ngokukhawuleza kunye nokuhlolwa kwe-E. coli O157: H7 ekutyeni, ukutya, iisampulu zamanzi kunye neesampuli zokusingqongileyo.
[Umgaqo wovavanyo]
Ngokomgaqo weteknoloji ye-PCR ye-fluorescent, iiprimers ezithile kunye ne-Taqman probes ziyilelwe uhlobo oluthile lwe-Escherichia coli O157: H7, kwaye ifunyenwe ngesixhobo se-PCR esinefluorescent, ukuze kuqatshelwe ukufunyanwa kwe-Escherichia coli O157: H7 Ukufunyanwa kwe-Quality ye-DNA.
Imixholo yeKit
Qaphela: Iprobe yesitishi se-ROX ayiqukwanga.
| Cabachasi | Inkcazo | Qubunyani |
| Buffer A | Umbhobho | 1 |
| Buffer B | Umbhobho | 1 |
| Ulawulo oluhle | Umbhobho | 1 |
| Ulawulo olubi | Umbhobho | 1 |
Usetyenziso olulindelekileyo
Isetyenziselwa ukufunyanwa ngokukhawuleza kunye nokuhlolwa kwe-E. coli O157: H7 ekutyeni, ukutya, iisampulu zamanzi kunye neesampuli zokusingqongileyo.
Iimeko zoGcino kunye noMhla wokuphelelwa
Gcina ku -20 ℃ ebumnyameni kwaye uphephe ukukhenkceza okuphindaphindiweyo kunye nokunyibilika.
Ixesha lokuqinisekisa li-12iinyanga, kwaye umhla wemveliso uboniswa kwipakethe yangaphandle.
Izixhobo kunye nezinto ezisetyenziswayo
Isixhobo se-PCR sobungakanani be-fluorescent, umpu wepipette kunye neengcebiso ezihambelanayo, i-vortex shaker, i-mini centrifuge.
Ukusetyenziswa
1. Isample Processing
1.1 Uhlobo lwesampulu: Le khithi ilungele ukutya, isondlo, iisampulu zamanzi kunye nezinye iisampuli ekurhaneleka ukuba zingcolisekile yi-Escherichia coli O157:H7.Kwiimveliso zenyama ezinzulu, iziphuzo kunye nezinye izinto eziqukethe i-pigment, kufuneka zihlanjululwe ukuze ziphephe ukuchaphazela ukuqokelela kwesignali ye-fluorescence.
1.2 Ukwenziwa kwesampuli: Jonga kwi "GB 4789.10-2016 yoKhuseleko loKutya uMgangatho weSizwe woKutya kweMicrobiological Examination ye-Escherichia coli O157: Uvavanyo lwe-H7" ukulungiselela iisampulu, inkcubeko yokutyebisa kunye nokuhlukaniswa kwe-Escherichia coli O157: H7.
- Nukutsalwa kwe-ucleic acid
Thatha i-20 mL yesisombululo sokutyebisa kwi-tube ye-1.5 mL ye-centrifuge, yongeza i-200 μL ye-microbial lysate (ikhithi eyongezelelweyo iyafuneka), i-vortex ye-30 imizuzwana, i-centrifuge ngokufutshane, kwaye ubeke bucala.
Amanqaku: Ukukhutshwa kwe-nucleic acid kwi-lysate kufuneka kugqitywe ngaphakathi kwemizuzu eyi-10, kwaye ayikwazi ukugcinwa ixesha elide.
3. Ukwandiswa kwe-Acid yeNucleic
3.1 Vula isixhobo se-fluorescent quantitative PCR ukuze sisetyenziswe.
I-Buffer A kunye ne-Buffer B ukusuka kwikhithi, zinyibilike ngokucokisekileyo, kunye ne-centrifuge ngokufutshane.Yongeza i-18 μL I-Buffer A kunye ne-2 μL ye-Buffer B kwityhubhu nganye yokusabela ye-PCR.Emva koko yongeza i-5 mL nganye yolawulo olubi, i-nucleic acid ekhutshweyo, kunye nolawulo oluhle kwi-PCR reaction tubes, i-cap the tubes, kunye ne-centrifuge ngokufutshane.
3.3 Dlulisa ityhubhu yokusabela kwe-PCR kumatshini we-PCR we-fluorescent, kwaye usebenzise ezi nkqubo zilandelayo ukwenza imifuniselo yokukhulisa : khetha i-25 mL yenkqubo yokusabela, qokelela izibonakaliso ze-fluorescence kwi-60 ° C kumjikelo ngamnye, kwaye ukhethe i-FAM yejelo lokubona.
| Inyathelo | Inkqubo | Inani lemijikelo |
| 1 | 37℃ 5min | 1 |
| 2 | 9 5 ℃ 3min | 1 |
| 3 | 95°C 15s | 4 0 |
| 60℃ 30s (qokelela i-fluorescence) |
Izikhokelo zohlalutyo lweengxaki
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。
Akukho RNA inokukhutshwa okanye isivuno se-nucleic acid siphantsi
Ngokuqhelekileyo zininzi izinto ezichaphazela ukusebenza kakuhle kokubuyisela, ezinje: umxholo we-RNA, indlela yokusebenza, umthamo we-elution, njl.
Uhlalutyo lwezizathu eziqhelekileyo:
1.I-ice bath okanye iqondo lokushisa eliphantsi (4 ° C) i-centrifugation ngexesha lokusebenza.
Ingcebiso: Ubushushu begumbi (15-25 ° C) ukusebenza, ungaze uhlambe umkhenkce kunye nobushushu obuphantsi be-centrifuge.
2. Ukugcinwa kwesampuli engafanelekanga okanye ukugcinwa kwesampuli ixesha elide.
Ingcebiso: Gcina iisampulu kwi-80 ° C okanye umkhenkce kwinitrogen engamanzi, kwaye ugweme ukusetyenziswa okuphindaphindiweyo komkhenkce;zama ukusebenzisa iisampulu ezisanda kuqokelelwa ukutsalwa kwe-RNA.
3.Isampula enganelanga i-lysis
Isincomo: Nceda uqinisekise ukuba isampuli kunye nesisombululo esisebenzayo (i-Linear Acrylamide) ixutywe ngokucokisekileyo kwaye ifakwe kwi-10 min kwindawo yokushisa (15-25 ° C)
4.I-eluent yongezwa ngokungalunganga
Ingcebiso: Qinisekisa ukuba i-ddH2O ye-RNase-Free yongezwa kumbindi wenwebu yekholamu yokucoca.
5.Umthamo ongafanelekanga we-ethanol ye-anhydrous kwi-Buffer viRW2
Ingcebiso: Nceda ulandele imiyalelo, yongeza umthamo ochanekileyo we-ethanol ye-anhydrous kwi-Buffer viRW2 kwaye uyixube kakuhle ngaphambi kokusebenzisa ikhithi.
6.Ukusetyenziswa kwesampulu engafanelekanga.
Iingcebiso: 200µl yesampulu nge-500μl ye-Buffer viRL.Umthamo wesampulu ogqithisileyo uya kukhokelela ekunciphiseni izinga lokutsalwa kwe-RNA.
7.Umthamo we-elution ongafanelekanga okanye ukukhutshwa okungaphelelanga.
Isiphakamiso: Umthamo ocacileyo wekholamu yokucoca yi-30-50μl;ukuba isiphumo asanelisi, kuyacetyiswa ukuba udibanise i-RNase-Free ddH eshushu ngaphambili.2O kunye nokwandisa ixesha lokubeka kwindawo yokushisa, njenge-5-10min
8.Ikholamu yokucoca inentsalela ye-ethanol emva kokuhlanjululwa kwi-Buffer viRW2.
Isiphakamiso: Ukuba i-ethanol isala emva kokuhlanjululwa kwi-Buffer viRW2 kunye ne-centrifugation ye-tube engenanto ye-2min, ikholamu yokucoca inokushiywa kwindawo yokushisa ye-5min emva kwe-tube centrifugation engenanto ukususa ngokupheleleyo i-ethanol eseleyo.
Ukonakaliswa kweemolekyuli ze-RNA ezihlambulukileyo
Umgangatho we-RNA ecocekileyo inxulumene nezinto ezifana nokugcinwa kwesampulu, ukungcoliseka kwe-RNase, kunye nokusebenza.
Uhlalutyo lwezizathu eziqhelekileyo:
1.Iisampulu eziqokelelweyo azigcinwanga ngexesha.
Ingcebiso: Ukuba isampulu ayisetyenziswanga ngexesha emva kokuqokelela, nceda uyigcine ku -80 ℃ okanye initrogen engamanzi ngoko nangoko.Xa kutsalwa iimolekyuli ze-RNA, zama ukusebenzisa iisampulu ezisanda kuqokelelwa xa kunokwenzeka.
2.Iisampulu eziqokelelweyo zazikhenkceza kwaye zinyibilike ngokuphindaphindiweyo.
Isiphakamiso: Gwema ukukhenkceza ngokuphindaphindiweyo kunye nokunyibilika (kungekho ngaphezu kweyodwa) ngexesha lokuqokelela isampuli kunye nokugcinwa, ngaphandle koko isivuno se-nucleic acid siya kuncipha.
3.I-RNase yaziswa kwigumbi lokusebenza okanye akukho ziglavu ezilahlwayo, iimaski, njl.njl.
Ingcebiso: Ukutsalwa kovavanyo lweemolekyuli ze-RNA kwenziwa ngcono kwigumbi lokusebenza elahlukileyo le-RNA, kwaye itafile yovavanyo iyacocwa phambi kovavanyo.Nxiba iiglavu ezilahlwayo kunye nemaski ngexesha lovavanyo ukunqanda ukuthotywa kwe-RNA okubangelwa kukwaziswa kwe-RNase.
I-4.I-reagent ingcolisekile yi-RNase ngexesha lokusetyenziswa.
Ingcebiso: Faka enye iViral RNA Isolation Kit kwimifuniselo ehambelanayo.
5.Ungcoliseko lwe-RNase yeetyhubhu ze-centrifuge, iingcebiso ze-pipette, njl. Ingcebiso: Qinisekisa ukuba iityhubhu ze-centrifuge, iingcebiso ze-pipette, kunye neepayipi zonke azina-RNase.
Iimolekyuli ze-RNA ezisulungekileyo zichaphazele iimvavanyo ezisezantsi
Iimolekyuli ze-RNA ezihlanjululwe yikholomu yokucoca ziya kuchaphazela iimvavanyo ezisezantsi ukuba kukho ii-ion zetyuwa ezininzi okanye iiprotheni, ezinje: ukuguqulelwa umva, i-Northern Blot, njl.
1.Kukho iiyoni zetyuwa ezishiyekileyo kwiimolekyuli ze-RNA eluted.
Isincomo: Qinisekisa ukuba umthamo ochanekileyo we-ethanol ye-anhydrous yongezwe kwi-Buffer viRW2, kwaye uhlambe ikholamu yokucoca kabini ngokwesantya esichanekileyo se-centrifugation kwimiyalelo yokusebenza; Ukuba kusekho i-ion yetyuwa eseleyo, unokongeza i-Buffer viRW2 kwikholamu yokucoca, kwaye uyishiye kwindawo yokushisa ye-5min.Emva koko yenza i-centrifugation ukususa i-ion yetyuwa ngowona mgangatho mkhulu
2.Kukho i-ethanol eshiyekileyo kwiimolekyuli ze-RNA
Isiphakamiso: xa uqinisekisa ukuba iikholomu zokucoca zihlanjululwe yi-Buffer viRW2, yenza i-centrifugation ye-tube engenanto ngokwesantya se-centrifugal kwimiyalelo yokusebenza.Ukuba kusekho i-ethanol eseleyo, inokushiywa imizuzu emi-5 kwiqondo lobushushu begumbi emva kokuba i-centrifugation yetyhubhu engenanto ukususa i-ethanol eseleyo ngowona mlinganiselo mkhulu.
Izikhokelo zoMyalelo:
I-Viral RNA Isolation Kit Manual Manual
