Cell Direct RT qPCR Kit—Taqman Direct Cell Lysis Cell Ready Mohato o le Mong qRT-PCR Kits Probe
Litlhaloso
Sehlahisoa sena se sebelisa mokhoa o ikhethileng oa lysis buffer ho lokolla ka potlako RNA ho tloha lisampole tsa sele e tsoetseng pele bakeng sa karabelo ea RT-qPCR, ho felisa ts'ebetso ea tlhoekiso ea RNA e jang nako le e boima, le metsotso e 7 feela ho fumana template e hlokahalang ea RNA, ka 5 × Direct RT Mix, 2 × Direct qPCR Mix-Taqman
5 × Direct RT Mix le 2 × Direct qPCR Mix-Taqman li na le mamello e matla ea inhibitor 'me li ka etsa phetoho e sebetsang hantle le ho hōlisa ka ho khetheha ho sebelisa lysate ea sampuli e lokelang ho lekanyetsoa e le template.Reagent e na le Foregene Reverse Transcriptase, Hot D-Taq DNA Polymerase, dNTPs, MgCl.2, Reaction Buffer, PCR Optimizer le Stabilizer, e ka sebelisoang le lysis buffer ho lemoha lisampole ka potlako le ha bonolo, 'me e na le litšobotsi tsa kutloisiso e phahameng, ho khetheha le botsitso.
Litlhaloso
200×20μl Rxns, 1000×20μl Rxns
Likarolo tsa Kit
| QuickEasyTMCell Direct RT-qPCR Kit-Taqman | ||||
| Likarolo tsa Kit 20μl qPCR Reaction System | DRT-01021 | DRT-01022 | Hlokomela | |
| 200 T | 1000 T | |||
|
Karolo I | Buffer CL | 4 ml | 20 ml | Lisele Lysis |
| Foregene Protease Plus II | 80 μl | 400 μl | ||
| Buffer ST | 400 μl | 1 ml × 2 | ||
|
Karolo II | DNA Raba | 80 μl | 400 μl | |
| 5×Direct RT Mix * | 160 μl | 800 μl | RT | |
| 2× Direct qPCR Mix-Taqman * | 1 ml × 2 | 1.7 ml × 6 | qPCR | |
| 20 × ROX Reference Dye | 40 μl | 200 μl | ||
| RNase-Free ddH2O | 1.7 ml | 10 ml | ||
| Buka ea Tataiso | 1 sengoathoana | 1 sengoathoana | ||
*:Cell Lysis, 5×Direct RT Mix, 2× Direct qPCR Mix-Taqman e ka rekoa ka thoko.
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.
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-Taqman 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.
Sehlomathiso1:Cle DirectRT-qPCR Karolo ea Kitt tlatsetso pakete
1.Cell Lysis Tharollo
| Seleng Lysis Tharollo | |||
| 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 |
| Motsoako oa RT | |
| 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 |
| qPCR Mix | ||
| Likarolo tsa Kit (Sistimi ea karabelo ea 20 μl) | DRT-01021-C1 | DRT-01021-C2 |
| 200 T | 1000 T | |
| 2 × Direct qPCR Mix-Taqman | 1 ml × 2 | 1.7 ml × 6 |
| 20 × ROX Reference Dye | 40 μl | 200 μl |
| RNase-Free ddH2O | 1.7 ml | 10 ml |
Litaelo:
