Qalabka Tooska ah ee Unugyada RT qPCR-SYBR GREEN I Toos Unugga Lysis Unug U Diyaarsan Qalabka qRT-PCR Hal-tallaabo
Sharaxaada
Xirmadani waxay adeegsataa nidaam u gaar ah oo lysis ah oo si dhakhso ah uga sii deyn kara RNA muunadaha unugyada dhaqameed ee falcelinta RT-qPCR, taas oo meesha ka saaraysa habka nadiifinta RNA waqti-qaadka iyo shaqada badan.Qaabka RNA waxa lagu heli karaa 7 daqiiqo oo kaliya.5-ta×Isku dhafka tooska ah ee RT iyo 2×Qalabka tooska ah ee qPCR Mix-SYBR ee uu qalabku bixiyo ayaa si degdeg ah oo wax ku ool ah u heli kara natiijooyinka PCR ee tirada dhabta ah.
5×Isku dhafka tooska ah ee RT iyo 2×Isku-dhafka tooska ah ee qPCR Mix-SYBR waxay leeyihiin dulqaad adag oo ka hortag ah, iyo lysate muunada waxaa loo isticmaali karaa qaab-dhismeedka RT-qPCR si toos ah.Qalabkani waxa uu ka kooban yahay RNA-ga gaarka ah ee xidhiidhka sare ee Foregene reverse transcriptase, iyo Hot D-Taq DNA polymerase, dNTPs, MgCl2, celiyaha falcelinta, PCR optimizer iyo stabilizer.
Tilmaamaha
200×20μl Rxns, 1000×20μl Rxns
Qaybaha kitka
| Qaybta I | Buffer CL |
| Foregene Protease Plus II | |
| Buffer ST | |
| Qaybta II | Tirtiraha DNA |
| 5× Toos ah RT Mix | |
| 2× Toos ah qPCR Mix-SYBR | |
| 50× ROX Tixraaca Midabka | |
| RNase-free ddH2O | |
| Tilmaamaha | |
Tilmaamaha & faa'iidooyinka
■Fudud oo wax ku ool ah: iyadoo la adeegsanayo tignoolajiyada Cell Direct RT, muunadaha RNA waxaa lagu heli karaa 7 daqiiqo oo keliya.
■ Baahida muunada waa yar tahay, maadaama ay yar tahay 10 unug ayaa la tijaabin karaa.
■ Soo saarista sare: waxay si dhakhso ah u ogaan kartaa RNA ee unugyada lagu beeray 384, 96, 24, 12, 6-cells.
■ Tirtiraha DNA-da si dhakhso ah ayuu u saari karaa genome-yada la sii daayay, si weyn ayuu u yareynayaa saameynta natiijooyinka tijaabada dambe.
■ Nidaamka RT iyo qPCR ee la hagaajiyay ayaa ka dhigaya laba-tallaabo dib-u-celinta RT-PCR mid hufan iyo PCR mid gaar ah, oo aad u adkeysi badan ka-hortagga falcelinta RT-qPCR.
Codsiga qalabka
Baaxadda codsiga: unugyada dhaqanka.
- RNA lagu sii daayay muunad lysis: kaliya lagu dabaqi karo qaabka RT-qPCR ee xirmadan.
-Qalabka waxa loo isticmaali karaa ujeedooyinka soo socda: falanqaynta muujinta hidde-sidaha, xaqiijinta saamaynta aamusinta hidde-sidaha ee siRNA, baadhista daroogada, iwm.
Jaantuska
Kaydinta iyo nolosha shelf
Qaybta I ee xirmadan waa in lagu kaydiyaa 4℃;Qaybta II waa in lagu kaydiyaa at -20 ℃.
Foregene Protease Plus II waa in lagu kaydiyaa 4℃, ha ku barafoobin -20 ℃.
Reagent 2×Tooska ah qPCR Mix-SYBR waa in lagu kaydiyaa -20℃gudcurka;haddii si joogto ah loo isticmaalo, waxa kale oo lagu kaydin karaa 4℃ kaydinta wakhtiga gaaban (ku isticmaal ilaa 10 maalmood gudahood).
Mabaadi'da naqshadaynta aasaasiga ah ee waqtiga dhabta ah PCR
Horudhac Horudhac iyo Horudhac Hordhac ah
PCR-da-waqtiga-dhabta ah, naqshadaynta asaasiga ahi aad bay muhiim u tahay.Furayaasha waxay la xiriiraan gaarnimada iyo hufnaanta kordhinta PCR, waxaana loo qaabayn karaa iyadoo la tixraacayo mabaadi'da soo socota:
Dhererka hore: 18-30bp.
Waxa ku jira GC: 40-60%.
Qiimaha Tm: Software-ka naqshadaynta aasaasiga ah, sida Primer 5, wuxuu ku siin karaa qiimaha Tm ee asaasiga ah.Qiimayaasha Tm ee hore iyo kuwa hoose waa inay ahaadaan kuwa ugu dhow ee suurtogalka ah.Qaaciddada xisaabinta Tm waxa kale oo la isticmaali karaa: Tm = 4 °C (G + C) + 2 °C (A + T).Marka la samaynayo PCR, heerkulka ka hooseeya qiimaha Tm ee aasaasiga ah ee 5 °C ayaa guud ahaan loo doortaa heer kulka soo jiidashada (kordhinta u dhiganta heerkulka xididada ayaa kordhin kara qeexida falcelinta PCR).
Qalabka aasaasiga ah iyo PCR:
Naqshadeynta asaasiga ah ee PCR dhererka alaabada ayaa la door bidaa 100-150bp.
Naqshadeynta aasaasiga ah ee aagga qaab dhismeedka sare ee template waa in laga fogaadaa sida ugu macquulsan.
Ka fogow samaynta 2 ama in ka badan oo saldhigyo dhammaystiran oo u dhexeeya 3′ daraf ee hore-u-socodyada sare iyo kuwa hoose.
Primer 3′ terminal saldhiga kuma jiri karo 3 dheeraad ah oo isku xigta G ama C.
Furayaasha laftoodu ma yeelan karaan dhismayaal is kaaba ah, haddii kale qaab-dhismeedka timaha ayaa la samayn doonaa, oo saamaynaya kor u qaadida PCR.
ATCG waa in loo qaybiyaa sida ugu siman ee suurtogalka ah ee isku xigxiga asaasiga ah, iyo 3′ saldhigga terminal waa in laga fogaadaa sida T.
Lifaaqa 1: Xidhmada kabka qaybta gacanta tooska ah ee RT-qPCR
1.Cell Lysis Solution
|
| |||
| Qaybaha kitka (24-hab lysis / ceel) | DRT-01011-A1 | DRT-01011-A2 | |
| 100 T | 500 T | ||
| QaybI | Buffer CL | 20 ml | 100 ml |
| Foregene Protease Plus II | 400 μl | 1 ml × 2 | |
| Buffer ST | 1 ml × 2 | 10 ml | |
| QaybII | Tirtiraha DNA | 400 μl | 1 ml × 2 |
2.RT Isku darka
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| |
| Qaybaha kitka (Nidaamka falcelinta 20 μl) | DRT-01011-B1 |
| 200 T | |
| 5× Toos ah RT Mix | 800 μl |
| RNase-free ddH2O | 1.7 ml × 2 |
|
| ||
| Qaybaha kitka (Nidaamka falcelinta 20 μl) | DRT-01011-C1 | DRT-01011-C2 |
| 200 T | 1000 T | |
| 2× Toos ah qPCR Mix-SYBR | 1 ml × 2 | 1.7 ml × 6 |
| 50× ROX Tixraaca Midabka | 40 μl | 200 μl |
| RNase-free ddH2O | 1.7 ml | 10 ml |
Buug-tilmaameedka:
Qalabka QuickEasyTM ee Tooska ah ee RT-qPCR Kit-SYBR Cagaaran I
