Qalabka Tooska ah ee Unugyada RT qPCR-Taqman Tooska ah Unugga Lysis Unug u diyaarsan Hal-tallaabo qRT-PCR Qalabka Baadhista
Sharaxaada
Alaabtani waxay isticmaashaa nidaam lysis u gaar ah si ay si dhakhso ah RNA uga sii deyso shaybaarada unugyada dhaqameed ee falcelinta RT-qPCR, baabi'inta habka nadiifinta RNA waqti-qaadka iyo shaqada badan, iyo kaliya 7 daqiiqo si loo helo qaabka RNA ee loo baahan yahay, oo leh 5 × Direct RT Mix, 2 × Direct qPCR Mix-Taqman oo ay bixiso xirmadu waxay si dhakhso ah oo hufan u heli kartaa PCR-waqtiga dhabta ah.
5× Direct RT Mix iyo 2× Direct qPCR Mix-Taqman waxay leeyihiin dulqaad adag oo ka hortag ah waxayna samayn karaan dib u habeyn hufan iyo xoojin gaar ah iyadoo la isticmaalayo lysate ee muunada si loo cabbiro qaab template ah.Reagent-ku waxa uu ka kooban yahay Foregene Reverse Transcriptase, Hot D-Taq DNA Polymerase, dNTPs, MgCl2, Reaction Buffer, PCR Optimizer iyo Stabilizer, kaas oo loo isticmaali karo lysis buffer si dhakhso ah oo sahlan loo ogaado shaybaarada, oo leh sifooyinka dareenka sare, gaar ahaaneed iyo xasiloonida.
Tilmaamaha
200×20μl Rxns, 1000×20μl Rxns
Qaybaha kitka
| Degdega fududTMQalabka Tooska ah ee RT-qPCR Kit-Taqman | ||||
| Qaybaha kitka 20μl qPCR Nidaamka Falcelinta | DRT-01021 | DRT-01022 | Ogow | |
| 200 T | 1000 T | |||
|
Qayb I | Buffer CL | 4 ml | 20 ml | Unugyada Lysis |
| Foregene Protease Plus II | 80 μl | 400 μl | ||
| Buffer ST | 400 μl | 1 ml × 2 | ||
|
Qayb II | Tirtiraha DNA | 80 μl | 400 μl | |
| 5× RT Isku dhafka tooska ah * | 160 μl | 800 μl | RT | |
| 2× Toos ah qPCR Mix-Taqman * | 1 ml × 2 | 1.7 ml × 6 | qPCR | |
| 20× ROX Tixraaca Midabka | 40 μl | 200 μl | ||
| RNase-free ddH2O | 1.7 ml | 10 ml | ||
| Buugga tilmaamaha | 1 gabal | 1 gabal | ||
*:Cell Lysis, 5× Direct RT Mix, 2× Direct qPCR Mix-Taqman si gooni gooni ah ayaa loo iibsan karaa
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.
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-Taqman 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.
Lifaaqa1:Ciyo DirectRT-qPCR Kit componenbaakad dheeri ah
1.Cell Lysis Solution
| 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 |
| RT Mix | |
| 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 |
| Isku dhafka qPCR | ||
| Qaybaha kitka (Nidaamka falcelinta 20 μl) | DRT-01021-C1 | DRT-01021-C2 |
| 200 T | 1000 T | |
| 2× Toos ah qPCR Mix-Taqman | 1 ml × 2 | 1.7 ml × 6 |
| 20× ROX Tixraaca Midabka | 40 μl | 200 μl |
| RNase-free ddH2O | 1.7 ml | 10 ml |
Buug-tilmaameedka:
