Mohloli oa fektheri oa China Rt PCR Assay Reagent bakeng sa Virus e Ncha ea 2019 (ORF1ab, N gene)
Ho nka mosebetsi o felletseng oa ho khotsofatsa litlhoko tsohle tsa bareki ba rona;ho fihlela tsoelopele e tsitsitseng ka ho bapatsa nts'etsopele ea bareki ba rona;hola ho ba molekane oa ho qetela oa tšebelisano-'moho ea bareki le ho holisa lithahasello tsa bareki bakeng sa mohloli oa fektheri oa China Rt PCR Assay Reagent bakeng sa Virus e Ncha ea 2019 (ORF1ab, N gene), Re itšepa hore re tla etsa likatleho tse ntle ha re ntse re le monyetla.Re ntse re labalabela ho ba e mong oa barekisi ba tšepahalang ba hau.
Ho nka mosebetsi o felletseng oa ho khotsofatsa litlhoko tsohle tsa bareki ba rona;ho fihlela tsoelopele e tsitsitseng ka ho bapatsa nts'etsopele ea bareki ba rona;hola ho ba molekane oa ho qetela oa tšebelisano 'moho oa bareki le ho holisa lithahasello tsa bareki bakeng saChina PCR Kit, Sesebelisoa sa ho lemoha PC, Re lebeletse, re tla tsamaisana le linako, re tsoele pele ho etsa lihlahisoa tse ncha.Ka sehlopha sa rona se matla sa lipatlisiso, lisebelisoa tse tsoetseng pele tsa tlhahiso, tsamaiso ea saense le litšebeletso tse phahameng, re tla fana ka lihlahisoa tsa boleng bo phahameng ho bareki ba rona lefatšeng ka bophara.Re u mema ka tieo hore u be balekane ba rona ba khoebo molemong oa bobeli.
Litlhaloso
Setsi sena se sebelisa sistimi e ikhethileng ea lysis buffer e ka lokollang RNA kapele ho lisampole tsa lisele tse hlahisitsoeng bakeng sa karabelo ea RT-qPCR, ka hona e felisa ts'ebetso ea tlhoekiso ea RNA e jang nako le e boima.Setšoantšo sa RNA se ka fumanoa ka metsotso e 7 feela.The 5×Direct RT Mix le 2×Direct qPCR Mix-SYBR reagents e fanoeng ke kit e ka fumana kapele le ka nepo liphetho tsa nako ea nnete tsa PCR.
5 × Direct RT Mix le 2 × Direct qPCR Mix-SYBR li na le mamello e matla ea inhibitor, 'me lysate ea lisampole e ka sebelisoa e le template ea RT-qPCR ka ho toba.Setsi sena se na le RNA e phahameng ea kamano e phahameng ea Foregene reverse transcriptase, le Hot D-Taq DNA polymerase, dNTPs, MgCl.2, buffer ea karabelo, PCR optimizer le stabilizer.
Litlhaloso
200×20μl Rxns, 1000×20μl Rxns
Likarolo tsa Kit
Karolo ea I | Buffer CL |
Foregene Protease Plus II | |
Buffer ST | |
Karolo ea II | DNA Raba |
5 × Direct RT Mix | |
2 × Direct qPCR Mix-SYBR | |
50 × ROX Reference Dye | |
RNase-Free ddH2O | |
Litaelo |
Likarolo le melemo
■ E bonolo ebile e sebetsa : ka theknoloji ea Cell Direct RT, lisampole tsa RNA li ka fumanoa ka metsotso e 7 feela.
■ Tlhokahalo ea sampole e nyane, e ka tlase ho lisele tse 10 li ka lekoa.
■ Phallo e phahameng: e khona ho lemoha ka potlako RNA liseleng tse entsoeng ka lipoleiti tse 384, 96, 24, 12, 6.
■ DNA Eraser e ka tlosa ka potlako liphatsa tsa lefutso tse lokollotsoeng, ea fokotsa haholo tšusumetso liphethong tse latelang tsa liteko.
■ Optimized RT le qPCR system e etsa hore mehato e 'meli ea RT-PCR reverse transcript e sebetse hantle le PCR e be e hlakileng haholoanyane, 'me e hanane le RT-qPCR reaction inhibitors.
Sesebelisoa sa Kit
Sekhahla sa ts'ebeliso: lisele tse tsoetseng pele.
- RNA e lokollotsoe ka mohlala oa lysis: e sebetsa feela ho template ea RT-qPCR ea kit ena.
- Thepa e ka sebelisoa bakeng sa merero e latelang: tlhahlobo ea polelo ea liphatsa tsa lefutso, netefatso ea siRNA-mediated gene silence effect, tlhahlobo ea lithethefatsi, joalo-joalo.
Setšoantšo
Storage le Shelf bophelo
Karolo ea I ea kit ena e lokela ho bolokoa ho 4 ℃;Karolo ea II e lokela ho bolokoa ho -20 ℃.
Foregene Protease Plus II e lokela ho bolokoa mochesong oa 4 ℃, e se ke ea hoama ho -20℃.
Reagent 2 × Direct qPCR Mix-SYBR e lokela ho bolokoa ho -20 ℃ lefifing;haeba e sebelisoa khafetsa, e ka boela ea bolokoa ho 4℃ bakeng sa polokelo ea nako e khutšoanyane (sebelisa pele ho matsatsi a 10). Nka mosebetsi o feletseng oa ho khotsofatsa litlhoko tsohle tsa bareki ba rona;ho fihlela tsoelopele e tsitsitseng ka ho bapatsa nts'etsopele ea bareki ba rona;hola ho ba molekane oa ho qetela oa tšebelisano-'moho ea bareki le ho holisa lithahasello tsa bareki bakeng sa mohloli oa fektheri oa China Rt PCR Assay Reagent bakeng sa Virus e Ncha ea 2019 (ORF1ab, N gene), Re itšepa hore re tla etsa likatleho tse ntle ha re ntse re le monyetla.Re ntse re labalabela ho ba e mong oa barekisi ba tšepahalang ba hau.
Mohloli oa fektheriChina PCR Kit, PCR Detection Kit, Re lebeletse, re tla tsamaisana le linako, re tsoele pele ho etsa lihlahisoa tse ncha.Ka sehlopha sa rona se matla sa lipatlisiso, lisebelisoa tse tsoetseng pele tsa tlhahiso, tsamaiso ea saense le litšebeletso tse phahameng, re tla fana ka lihlahisoa tsa boleng bo phahameng ho bareki ba rona lefatšeng ka bophara.Re u mema ka tieo hore u be balekane ba rona ba khoebo molemong oa bobeli.
Melao-motheo ea moralo oa nako ea 'nete ea PCR
Pele ho Primer le Reverse Primer
Bakeng sa nako ea 'nete ea PCR, moralo oa primer o bohlokoa haholo.Li-primers li amana le ho hlaka le katleho ea ntlafatso ea PCR, 'me li ka etsoa ho ipapisitsoe le melaoana e latelang:
Bolelele ba pele: 18-30bp.
Likahare tsa GC: 40-60%.
Tm boleng: Software ea moralo oa Primer, joalo ka Primer 5, e ka fana ka boleng ba Tm ba primer.Litekanyetso tsa Tm tsa li-primers tse holimo le tse tlase li lokela ho ba haufi ka hohle kamoo ho ka khonehang.Mokhoa oa ho bala oa Tm o ka boela oa sebelisoa: Tm = 4 °C (G + C) + 2 °C (A + T).Ha o etsa PCR, mocheso o ka tlase ho boleng ba pele ba Tm oa 5 ° C hangata o khethoa joalo ka mocheso o kenang (keketseho e lumellanang ea mocheso oa annealing e ka eketsa boits'oaro ba PCR).
Li-Primers le lihlahisoa tsa PCR:
Bolelele ba sehlahisoa sa ntlafatso ea PCR ka ho khetheha ke 100-150bp.
Li-primer tsa moralo sebakeng sa bobeli sa sebopeho sa template li lokela ho qojoa ka hohle kamoo ho ka khonehang.
Qoba ho theha metheo e 2 kapa ho feta e tlatselletsanang lipakeng tsa 3′ ea lithako tse holimo le tse tlase.
Primer 3′ terminal base e ka se be teng ka tse ling tse 3 tse latellanang tsa G kapa C.
Li-primers ka botsona li ke ke tsa ba le meaho e tlatselletsang, ho seng joalo sebopeho sa hairpin se tla thehoa, se amang ntlafatso ea PCR.
ATCG e lokela ho ajoa ka ho lekana ka hohle kamoo ho ka khonehang ka tatellano ea pele, 'me setsi sa 3′ se lokela ho qojoa joalo ka T.
Sehlomathiso sa 1: Sephutheloana sa tlatsetso sa Cell Direct RT-qPCR Kit
1.Seleng Lysis Tharollo
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Likarolo tsa Kit (Sistimi ea lysis ea seliba tse 24 / seliba) | DRT-01011-A1 | DRT-01011-A2 | |
100 T | 500 T | ||
KaroloI | Buffer CL | 20 ml | 100 ml |
Foregene Protease Plus II | 400 μl | 1 ml × 2 | |
Buffer ST | 1 ml × 2 | 10 ml | |
KaroloII | DNA Raba | 400 μl | 1 ml × 2 |
2.RT Motsoako
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Likarolo tsa Kit (Sistimi ea karabelo ea 20 μl) | DRT-01011-B1 |
200 T | |
5 × Direct RT Mix | 800 μl |
RNase-Free ddH2O | 1.7 ml × 2 |
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Likarolo tsa Kit (Sistimi ea karabelo ea 20 μl) | DRT-01011-C1 | DRT-01011-C2 |
200 T | 1000 T | |
2 × Direct qPCR Mix-SYBR | 1 ml × 2 | 1.7 ml × 6 |
50 × ROX Reference Dye | 40 μl | 200 μl |
RNase-Free ddH2O | 1.7 ml | 10 ml |