Theko e kholo ea China High Sensitivity One-step Probe Rt-Qpcr Kit V2
Tlhaloso ea Kit:
◮ 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.
◮Ho sebetsa haholo: e ka lemoha ka potlako RNA liseleng tse hlahisitsoeng ka lipoleiti tse 384, 96, 24, 12, 6.
◮DNA Eraser e ka tlosa liphatsa tsa lefutso tse lokollotsoeng kapele, ea fokotsa haholo tšusumetso liphethong tse latelang tsa liteko.
◮Sistimi e ntlafalitsoeng ea RT le qPCR e etsa hore khatiso e khutlisetsang mehato e 'meli e sebetse hantle le PCR e tobane le ho feta, hape e hanyetsane le RT-qPCR reaction inhibitors.
Re bile moetsi ea nang le phihlelo.Ho hapa bongata litifikeiting tsa bohlokoa tsa 'maraka oa eona bakeng sa theolelo e kholo ea China High Sensitivity One-Step Probe Rt-QpcrKit V2, Boleng ke bophelo ba feme, Tsepamisa maikutlo ho tlhoko ea bareki ke mohloli oa ho phela le nts'etsopele ea k'hamphani, Re khomarela botšepehi le boikutlo bo botle ba ho sebetsa ka tumelo, re lebeletse ho tla ha hao!
Re bile moetsi ea nang le phihlelo.Ho hapa bongata litifikeiting tsa bohlokoa tsa 'maraka oa eona bakeng saChina Taq DNA Polymerase, Qpcr, Khampani ea rona e tšepisa: litheko tse utloahalang, nako e khutšoanyane ea tlhahiso le tšebeletso e khotsofatsang ka mor'a ho rekisa, re boetse re u amohela ho etela fektheri ea rona ka nako leha e le efe eo ue batlang.Ke lakatsa eka joale re na le khoebo e monate le e telele hammoho !!!
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 nakong ea matsatsi a 10) .Re bile moetsi ea nang le phihlelo.Ho hapa bongata litifikeiting tsa bohlokoa tsa 'maraka oa eona bakeng sa theolelo e kholo ea China High Sensitivity One-Step Probe Rt-Qpcr Kit V2, boleng ke bophelo ba feme, Tsepamisa maikutlo ho tlhokahalo ea bareki ke mohloli oa ho phela le nts'etsopele ea k'hamphani, Re khomarela botšepehi le boikutlo bo botle ba ho sebetsa ka tumelo, re lebeletse ho tla ha hao!
Theko e kholoChina Taq DNA Polymerase, Qpcr, Khamphani ea rona e tšepisa: litheko tse utloahalang, nako e khutšoanyane ea tlhahiso le tšebeletso e khotsofatsang ka mor'a ho rekisa, re boetse re u amohela ho etela fektheri ea rona ka nako leha e le efe eo ue batlang.Ke lakatsa eka joale re na le khoebo e monate le e telele hammoho !!!
QuickEasyTM Cell Direct RT-qPCR Kit -Taqman
Katse.No.DRT-01021/01022
Bakeng sa lisele tse tobileng tsa RT-qPCR tse sebelisang ≤ 1000,000 lisele
Kenyelletso ea sehlahisoa
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.
Likarolo tsa sehlahisoa
Theknoloji e bonolo, e sebetsang ea Cell Direct RT e nkang metsotso e 7 feela ho fumana lisampole tsa RNA.
Litlhoko tsa lisampole li nyane, 'me bonyane lisele tse 10 tse hlahisitsoeng li ka sebelisoa bakeng sa liteko.
Phaello e phahameng ea ho fumana RNA ka potlako ea lisele tse tsoetseng pele joalo ka lipoleiti tse 384, 96, 24, 12 le 6 tsa seliba.
DNA Eraser e khona ho tlosa ka potlako liphatsa tsa lefutso tse lokollotsoeng, ho fokotsa haholo tšusumetso liphethong tse latelang tsa liteko.
Sistimi e ntlafalitsoeng ea RT le qPCR e thusa RT-PCR ea mehato e 'meli e nang le phetisetso e hlakileng haholoanyane, e ikhethang, le mamello e matla ea RT-qPCR reaction inhibitor.
Sesebelisoa sa Kit
Sekhahla sa ts'ebeliso: Lisele tse hlophisitsoeng.
Sample lysis e tolokile RNA: e sebelisoa feela e le template ea RT-qPCR ea mehato e 'meli.
Li-Kits li ka sebelisetsoa merero e latelang: tlhahlobo ea polelo ea taolo ea liphatsa tsa lefutso, tlhahlobo ea allele, tlhahlobo ea lithethefatsi, jj.
Mefokolo ea Kit
Likhechana tse matlafalitsoeng ≤ 300 bp.
Li-kits li sebelisetsoa lisele tse ncha tsa setso.
Taolo ea boleng ba sehlahisoa
Ho latela FOREGENE's Total Quality Management System, beche ka 'ngoe ea Cell Direct RT-qPCR series kits e lekoa ka matla makhetlo a mangata ho netefatsa ho ts'epahala le botsitso ba boleng ba beche ka 'ngoe ea lisebelisoa.
Likahare tsa Kit
| QuickEasyTM Cell Direct RT-qPCR Kit-Taqman | ||||
| Likarolo tsa Kit20μl 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, lintlha li fanoe ho Sehlomathiso sa 1 (Leqephe la 13).
Maemo a polokelo
1. Maemo a ho Romela
Ts'ebetso eohle ea lipalangoang tse tlase tsa mocheso o tlase oa leqhoa, ho netefatsa hore khiti e maemong a <4 °C.
2. Maemo a polokelo
Boloka karolo ea I ho 4°C le Karolo ea II ho -20°C.
Foregene Protease Plus II e lokela ho bolokoa ka mocheso oa 4°C, e seng ho hoama ho -20°C.
Reagent 2 × Direct qPCR Mix-Taqman e bolokiloe ho -20 ° C, kapa ho 4 ° C bakeng sa tšebeliso ea nako e khutšoanyane haeba e sebelisoa hangata (ka matsatsi a 10).
Lintlha tsa karolo ea Kit
Buffer CL: E fana ka tikoloho e hlokehang bakeng sa karabelo ea lisele.
Buffer ST: E felisa ntho e sebetsang ho lysate ho qoba litlamorao ho RT e latelang.
DNA Eraser: DNA remover, phello ea ho tlosa genome litekong tse latelang.
5 × Direct RT Mix: E na le kamano e phahameng ea RNA Foregene Reverse Transcriptase, RNase Inhibitor, dNTPs, stabilizers, enhancers, optimizers, le li-primer tsa khatiso tse khutlisetsang morao bakeng sa ho lumellana hantle (Random Primer, Oligo(dT)18Sethome).
Foregene Protease Plus II: Boemong ba lysis buffer, lisele li lysed ho lokolla nucleic acid.
2 × Direct qPCR Mix-Taqman: Reagent ena e na le Hot D-Taq DNA Polymerase, dNTPs, MgCl2, buffer ea karabelo, PCR optimizer, le stabilizer.
20 × ROX Reference Dye: Ka kakaretso e sebelisoa ho lisebelisoa tsa amplification tsa Real Time PCR tsa ABI, Stratagene le lik'hamphani tse ling, e sebelisetsoa ho lokisa phapang pakeng tsa li-tubes tsa PCR le li-tubes tse bakoang ke liphoso tsa dosing tsa PCR.Khokahano ea 20 × ROX Reference Dye e hlokehang bakeng sa lisebelisoa tse fapaneng e fapane, 'me mosebelisi a ka e eketsa ho latela mohopolo o khothaletsoang oa sesebelisoa.
RNase-Free ddH2O: Metsi a hloekileng a se nang RNase a se nang metsi a hloekileng bakeng sa karabelo ea mehato e 'meli ea RT-qPCR.
Mehato ya tlhokomelo:(Etsa bonnete ba hore u bala litemoso ka hloko pele u sebelisa kit)
Ela hloko mokhoa oa ts'ebetso oa teko ho qoba tšilafalo pakeng tsa lisampole.
Ela hloko bohloeki ba tikoloho ea liteko le lisebelisoa ho qoba tšilafalo ea RNase le ho senyeha ha RNA.
Nka lisampole tse ncha kapa tse bolokiloeng hantle 'me u se ke ua sebelisa lisampole tsa lisele tse hatselitsoeng khafetsa.
5 × Direct qPCR Mix, 2× Direct qPCR Mix-Taqman e lokela ho qoba ho qhibiliha khafetsa, ho seng joalo e tla ama phetisetso ea morao-rao le ts'ebetso ea PCR.
Prepalijo tsa lijopelets'ebetso
Etsa bonnete ba hore u bala litaelo ka hloko pele u sebelisa setsi sena.Cell Direct RT-qPCR Kit e bonolo, e bonolo ebile e potlakile ho sebetsa, 'me litaelo li fana ka leseli le felletseng mabapi le khiti eohle le mokhoa oa ho e sebelisa ka nepo.Ka kopo, lokisa lisebelisoa tse hlokahalang tsa liteko le lisebelisoa pele u li sebelisa.
Lisebelisoa le lisebelisoa tsa liteko
◆ Lisele tsa setso.
◆ 1.5 ml kapa 2 ml, RNase-/DNase-Free centrifuge tube, RNase-/DNase-Free tip, 0.2 ml sterile qPCR tube.
◆ mochini oa qPCR, pipette, tabletop centrifuge (≥13,400×g) (ho ipapisitse le litlhoko tsa liteko), joalo-joalo.
Polokeho
◆ Sehlahisoa sena ke sa merero ea lipatlisiso tsa saense feela, ka kopo u se ke ua e sebelisa bakeng sa merero ea meriana, ea bongaka, ea lijo le ea litlolo.
◆ Ha u sebelisa lik'hemik'hale, apara liaparo tse loketseng tsa laborateng, liatlana, likhalase tse sireletsang, joalo-joalo.
Tshebetsobatataisi
Litsamaiso tsa Cell Lysis, RT systems, le qPCR reaction supplement packs li ka rekoa ka thoko, bakeng sa lintlha tse ho Appendix 1 (LEQEPHE 13).
Tataiso ea ts'ebetso
A: Mohlala oa tlhahiso ea RNA
1.Lisele li ne li entsoe esale pele: Hlatsoa poleiti ea setso sa sele ka PBS e batang, ebe u lisa lisele (10-10).6), 106 ho feta palo ea lisele, ho khothaletsoa Foregene the Cell an RNA Isolation Kit the Total (DE-03111) kapa Animal Total RNA Isolation Kit (DE-03011) bakeng sa ho ntša RNA le tlhoekiso.
1.1.Lisele tse khomarelang (mohlala oa 24- seliba)
1.1.1.Fumana palo ea lisele selibeng ka seng, etsa qeto ea hore palo ea lisele ke 1 × 105, 'me u sebelise pipette ho tlosa setso sa setso ho tsoa sejana sa setso.
1.1.2.Kenya 200 μl ea 1 × PBS e hatselitsoeng pele selibeng ka seng.Se ke oa letsa peipi khafetsa 'me u tlose PBS lilibeng.Sekama poleiti 'me u tlose PBS e ngata kamoo ho ka khonehang.Tsoela pele ho mohato oa 2.
1.1.3.Sejo se fapaneng sa setso sa sele kapa tafole ea nomoro ea litšupiso 1-1 ka sejana sa setso sa sele se ile sa eketsoa se pholile 1 × PBS bakeng sa ho hlatsoa lisele.
Letlapa1-1: Tekanyo ea PBS bakeng sa lipalo tse fapaneng tsa lisele
| Setso mofuta oa poleiti | Palo ea lisele / seliba | 1 × PBS/ hantle |
| 6 - hantle | 1 × 106 | 1000 μl |
| 12 - hantle | 2 × 105 | 400 μl |
| 24 - hantle | 105 | 200 μl |
| 96 - hantle | 104 | 50 μl |
| 384 - hantle | 5 × 103 | 25 μl |
Hlokomela:Ho etsa bonnete ba hore ho na le lisele tse khomarelang tse tiileng,palo e kholo ea tahlehelo ea lisele e qojoang ha ho hlatsuoa .
1.2.Lisele tse emisitsoeng kapa lisele tse khomarelang tse hlahisitsoeng ka lipoleiti tse se nang porous
1.2.1.Lisele tse khomaretsoeng tse hlahisitsoeng ka lipoleiti tsa seliba tse se nang li-multi-multiple (lisele tse emisitsoeng li qala ho tloha mohatong o latelang oa 1.2.2), li bokella le ho arola lisele ho ea ka mokhoa o tloaelehileng oa ho bokella lisele, ebe o li beha ka poleiti ea setso kapa tube ea centrifuge;haeba trypsinization e sebelisoa, e hloka centrifugation ho bokella lisele le ho tlosa trypsin e setseng, e ekelitse lisele tse tsosolositsoeng tsa PBS ho lisele ka bomong ho qhala lisele.
1.2.2.Kamora palo ea lisele tse baloang, lisele tse qotsitsoeng 1 × 105 e le 'ngoe ho li-tubes tsa centrifuge, bokella lisele ka centrifugation ho 1000 × g bakeng sa 10 min.
1.2.3.Eketsa 200 μl PBS ho tube ea centrifuge, u se ke ua letsa pipe hangata, 'me u labalabela PBS ka ho toba.tsoela pele ho mohato oa 2. (Haeba ho le thata ho fokotsa 'me lisele li tsosolosoa hape, li ka' na tsa etsoa 1000 × g centrifuged 10 min ka mor'a ho lahla matla a maholo, pellet ea sele e tsoela pele mohato oa 2)
2.Cell lysis: Tlosa Buffer CL, mocheso oa eona o lekanang le mocheso oa kamore, DNA Eraser le Foregene Protease Plus II, ho latela tafole e latelang 1-2 e lokiselitsoeng lysis system: (Tharollo ea Lysis e loketse ho sebelisoa).
Letlapa la 1-2: cleavage ho lokisoa ha sistimi (Hlokomela: ho lokisoa holim'a leqhoa)
| Karolo (Cell Lysis Master Mix) | 6-lijana tsa seliba | 12-seliba poleiti | 24-seliba poleiti | 96-seleba sejana | 384-seliba sejana |
| 1000 μl / hantle | 400 μl / hantle | 200 μl / hantle | 50 μl / hantle | 25 μl / hantle | |
| Buffer CL | 960μl | 384μl | 192μl | 48μl | 24μl |
| DNA Raba | 20μl | 8μl | 4μl | 1μl | 0.5 μl |
| Foregene Protease Plus II | 20μl | 8μl | 4μl | 1 μl | 0.5 μl |
3.( 24 -well plate e le mohlala) Pipette 200 μl ea cell lysis master mix ka har'a seliba se seng le se seng, Bulela khafetsa ka makhetlo a 5-10, E kenye mocheso oa kamore (20-25 ℃) metsotso e 5.
Hlokomela:Ho qoba ho thehoa ha li-bubble, ka kopo ha sekala sa pipette sa pipette se fetotsoe ho 200μl kapa ka tlase.Lisele li ka 'na tsa bonahala li le maru ka mor'a lysis, e leng ntho e tloaelehileng.
4. (24 -plate ea hantle e le mohlala) e kenngoa ka mokelikeli 20 μl Buffer ST (litsamaiso tse fapaneng tsa lysis Buffer ST e kenyelelitsoe ka chelete e bontšitsoeng ho Lethathamo la 1-3), pipetting e pheta-phetoang ka makhetlo a 5-10, mocheso oa kamore (20-25 ℃) e ne e kenngoa ka 2 min.
Hlokomela:Nōka ea pipette e lahliloe ka tlase holimo, ho netefatsa hore lysate e ekelitsoe,ho qoba ho thehoa ha li-bubble, ka kopo ha sekala sa pipette sa pipette se fetotsoe ho 200μl kapa ka tlase.
Lethathamo la 1-3:Kenya Buffer ST
| Buffer ST | 6 - poleiti ea seliba | 12- seliba sejana | 24- seliba sejana | 96- seliba sejana | 384- seliba sejana |
| 100 μl / hantle | 40 μl / hantle | 20 μl / hantle | 5 μl / hantle | 2.5 μl / hantle |
5.Lysate e sebelisetsoa liteko tse latelang tsa RT-qPCR.Haeba liteko tse latelang li ke ke tsa etsoa ka nako, ka kopo e boloke holim'a leqhoa ka nako e fetang 2hr, 'me u boloke ho -20 ℃ kapa -80 ℃ (ho feta likhoeli tse tharo).
B: Tokisetso ea tsamaiso ea RT
1.Nka 5 × Direct RT Mix 'me ue behe holim'a bate ea leqhoa, e lumelle hore e qhibilihe ka tlhaho,' me ka bonolo e kopanye bakeng sa tšebeliso ea morao;ntša RNase-Free ddH2O 'me u e qhibilihe ebe u e beha holim'a bate ea leqhoa hore e sebelisoe hamorao.Lokisetsa mokhoa oa ho arabela holim'a leqhoa ho ea ka Lethathamo la 2-1 ka tlase.
Letlapa la 2-1: Tokisetso ea tsamaiso ea RT
| Sistimi ea RT e eketsa litaba | Ka chelete | Khatiso ea ho qetela | |
| 5 × Motsoako o tobileng oa RT | 4μl | 8 μl | 1 × |
| Cell Lysates (Setšoantšo sa RNA) | 4 μl | 8 μl | Eketsa phetoho ea sebaka (10 -40%) |
| RNase-Free ddH2O | 12 μl | 24 μl | - |
| Kakaretso ea Bolumo | 20 μl | 40 μl | - |
2.Ka mor'a ho phethoa ha tsamaiso ea tsamaiso, ka bonolo e kopantsoe le centrifuged ka bokhutšoanyane tafoleng e latelang 2 -2 reaction conditions RT reaction.
Letlapa la 2-2: Boemo ba boemo ba RT Reaction
| Mohato | Mocheso | nako | dikahare |
| 1 | 42 °C | 15-30 mets | cDNA synthesis |
| 2 | 95 °C | 5 mets | Inactivated reverse transcriptase |
| 3 | 4 °C | N/A |
3.Ka mor'a ho phethoa ha karabelo, sehlahisoa sa karabelo se ile sa behoa ka ho toba holim'a leqhoa bakeng sa qPCR, ka kopo, beha polokelo ea nako e telele -20 ℃ kapa -80 ℃.
Tlhokomeliso: Ka lebaka la ts'ebeliso ea thempleite e sa hloekisoang, meea e tšoeu e ka hlaha sehlahisoang sa mongolo oa reverse.Ena ke ntho e tloaelehileng.Centrifuge supernatant hang-hang bakeng sa liteko tse latelang.
Tharollo e hlahisoang ke RT e eketsoa ho Sistimi e latelang ea Karabelo ea Nako ea 'Nete ea PCR, ho khothaletsoa ho eketsa lipalo ho tloha ho 10-30% ea sistimi ea karabelo.
C: Tokiso ea sistimi ea karabelo ea qPCR
1.Palo e nepahetseng ea B e lokiselitsoeng mohatong oa template ea cDNA ho latela tafole e latelang 3-1 ho lokisa mokhoa oa ho arabela.
Tlhokomeliso: Palo ea template ea cDNA e nka 10-30% ea sistimi ea qPCR.Ka mohlala, tsamaisong ea 20μl qPCR, eketsa 2-6 μl ea lysis buffer, empa eseng ho feta 6 μl.
2.The optimization maemo a matle a qPCR (Annealing mocheso, joalo-joalo) bakeng sa karabelo ea qPCR (Maemo a karabelo a fanoeng ho Lethathamo la 3-2).
Tlhokomeliso: Leka ho sebelisa maemo a ntlafalitsoeng bakeng sa karabelo ea qPCR ho fumana liphetho tse ntle.
Letlapa la 3-1: Tokisetso ea mokhoa oa ho arabela oa PCR
| Sistimi ea RT e eketsa litaba | Ka chelete | Khatiso ea ho qetela |
| 2 × Direct qPCR Mix-Taqman | 10 μl | 1× |
| Primer ea pele (10μM) | 0.4 μl | 50-900 nM 1* |
| Reverse Primer (10μM) | 0.4 μl | 50-900 nM 1* |
| Probe(10μM) | 0.2 μl | 200nM |
| template ea cDNA (e fumanoe mohato oa B) | 4 μl | 10-30% |
| RNase-Free ddH2O | - | |
| 20×ROX Reference Dae 3* | - | - |
| Kakaretso ea Bolumo | 20 μl |
1 *: Khatiso ea Primer e ka fetoloa maemong a 50-900 nM ha ts'ebetso ea karabelo ea primer e fokola.
Tlhokomeliso: Sistimi ea qPCR e ka fetoloa ho latela litlhoko tsa liteko le mofuta oa sebaesekele sa fluorescence.Bakeng sa qPCR ka 50μl, fetola litekanyetso tsa reagent ka ho lekana ho latela 20μl tsamaiso.
| Mochini oa PCR oa nako ea 'nete | ROX Reference Dye mahloriso a ho qetela |
| ABI PRISM7000/7300/7700/7900HT/Mohato oa Pele, joalo-joalo. | 1×(mohlala. 20 μl system, eketsa 1 μl 20×ROX Reference Dye) |
| ABI 7500/7500 Fast and StratageneMx3000P/Mx3005P/Mx4000, joalo-joalo. | 0.5×(mohlala. 20 μl tsamaiso, eketsa 0.5 μl 20×ROX ReferenceDye) |
2 *: Khetha motsoako o nepahetseng oa ho qetela oa ROX Reference Dye ho latela fluorescence quantitative thermal cycler.Lintlha tse nepahetseng ka ho fetesisa tsa ROX Reference Dye bakeng sa libaesekele tse tloaelehileng tsa fluorescence li bonts'oa tafoleng e ka tlase:
Lethathamo la 3-2: maemo a karabelo ea qPCR a fanoa
| Mehato e 'meli | Mocheso | Nako | Lipotoloho | Litaba |
| 1 | 95℃ | 3 mets | 1 | Boikemisetso ba tlhaho |
| 2 | 95℃ | 5-10 sec | 40 | Setšoantšo sa denaturation |
| 3 | 60-65 ℃ | 20-30 sec | Anealing/ Ketsetso |
Tlhokomeliso: Bakeng sa ho fumana phello e ntle ka ho fetisisa ea qPCR, gradient PCR e ka sebelisoa ho ntlafatsa maemo a karabelo bakeng sa litempele tse fapaneng le li-primers tse fapaneng.Maemo a karabelo ea PCR a fapana ho ipapisitse le analyzer ea fluorescence, template, primer, joalo-joalo. Ts'ebetsong e ikhethileng, maemo a nepahetseng a karabelo a hloka ho raloa ho latela maemo a khethehileng a fluorescence quantitative thermal cycler, mofuta oa template, boholo ba sekhechana sa thahasello, tatellano ea motheo ea sekhechana se holisang, bolelele ba nako, jj.
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 Primer: 18-30bp.
◆ Likahare tsa GC: 40-60%.
◆ Boleng ba Tm: 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).
◆ Primers le lihlahisoa tsa PCR:
◆ Design primer PCR amplification sehlahisoa ka ho khetheha 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 thehoa ha metheo ea 2 kapa ho feta e tlatsanang pakeng tsa 3' lipheletsong tsa li-primers tse holimo le tse tlaase.
◆ Primer 3′ terminal base e ka se be teng le 3 tse eketsehileng tse latellanang tsa G kapa C.
◆ Li-primers ka botsona li ke ke tsa e-ba le mehaho e tlatsetsang, ho seng joalo mohaho oa moriri o tla thehoa, o ama PCR amplification.
◆ ATCG e lokela ho ajoa ka mokhoa o lekanang 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
|
| |||
| 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 |
|
| |
| 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 |
Motsoako oa 3.qPCR
|
| ||
| 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 |
Foregene ea Lefatše
Foregene Co., Ltd
Mohala: 028-83360257,028-83361257
E-mail ：info@foregene.com
Http://www.foregene.com
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