Cell Direct RT qPCR Kit—SYBR GREEN I Direct Cell Lysis Cell Ready Mohato o le Mong qRT-PCR Kits
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.Ea 5×Direct RT Mix le 2×Li-reagents tsa Direct qPCR Mix-SYBR tse fanoeng ke kit li ka fumana kapele le ka nepo liphetho tsa nako ea 'nete tsa PCR.
5×Direct RT Mix le 2×Direct qPCR Mix-SYBR e na le mamello e matla ea inhibitor, 'me lysate ea lisampole e ka sebelisoa e le template ea RT-qPCR ka kotloloho.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 ho 4℃, e seke ea hatsele ho -20 ℃.
Reagent 2×Direct qPCR Mix-SYBR e lokela ho bolokoa ho -20℃lefifing;ha e sebelisoa khafetsa, e ka bolokoa ho 4℃ bakeng sa polokelo ea nako e khuts'oane (sebelisa nakong ea matsatsi a 10).
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 |
Litaelo:
