(96-si fiican) Qalabka QuickEasy Cell Tooska ah ee RT-qPCR - SYBR Cagaaran I
Sharaxaada
The(96-si fiican) QuickEasyTM Qalabka Tooska ah ee Unugyada RT-qPCR-SYBR Cagaaran Iwaxay bixisaahab u gaar ah lysis u kaydiyato si degdeg ah u sii daaya RNAunug dhaqanmuunado loogu talagalay falcelinta RT-qPCR, baabi'inta wakhtiga iyo shaqada badanHabka nadiifinta RNA, oo kaliya waxay qaadataa 7 daqiiqo si loo helo qaabka RNA ee loo baahan yahay.The5× Toos ah RT Mixiyo2× Toos ah qPCR Mix-SYBRlagu bixiyo sanduuqa si degdeg ah iyosi wax ku ool ah u hel tirada waqtiga-dhabta ahNatiijooyinka PCR
5× Direct RT Mix iyo 2× Direct qPCR Mix-SYBR waxay leeyihiin dulqaad adag oo ka hortag ah, waxayna isticmaali karaan lysate ee muunada si loogu tijaabiyo qaab-qoraal hufan iyo kordhin gaar ah.Reagent-ku waxa uu ka kooban yahay Foregene u gaar ah Reverse Transcriptase oo leh xidhiidh sare oo RNA ah, iyo sidoo kale Hot D-Taq DNA Polymerase, dNTPs, MgCl2 , falcelinta falcelinta, PCR optimizer iyo stabilizer , waxaana loo isticmaali karaa iyada oo lala kaashanayo lysis buffer si loo ogaado shaybaarka si dhakhso ah, si sahlan oo sax ah, waxayna leedahay sifooyinka dareenka sare, gaar ahaan xoog leh iyo xasilooni wanaagsan.
Xirmada waxaa loogu talagalay lysis micro-system lysis of 96-unugyada si fiican u dhaqan, oo uu leeyahay labis wanaagsan iyo joogtayn;Qaybaha xirmada waxay bixiyaan falcelinta 96 lysis, 96 falcelinta qoraalka dib u celinta iyo falcelinta 96 × 2 qPCR, kaas oo la kulmi kara saxanka unugyada 96-si fiican ee hal mar la isticmaalo, isaga oo iska ilaalinaya wasakheynta ay keento furitaanka soo noqnoqda, qaboojinta iyo dhalaalinta reagents iyo hoos u dhaca waxqabadka reagent.
Qaybaha kitka
| Halabuurka Kit ( 50 μL lysis system/20 μLRT habka falcelinta /20μLqPCR habka falcelinta) | DRT-03011 | Odhaah | |
| 96T | |||
| QaybI | BufferCL | 5 ml | UnugLysis |
| ForegeneProtease Plus II | 100 μL | ||
| BufferST | 500 μL | ||
| Qayb II | DNATirtir | 100 μL | |
| 5× Toos ah RT Mix | 400 μL | RT | |
| 2× Isku dhafka tooska ah ee qPCR-SYBR | 1 mL × 2 | qPCR | |
| 50× ROX Tixraaca Midabka | 400µL | ||
| RNase- BilaashddH2 O | 1.7ml |
| |
| Msannadkii | 1 gabal | 1 adeeg | |
*: Tixaha DNA-da reagent-ka ee lysis waxa uu ku jiraa Qaybta II ee xirmada;Unugyada Lysis, RT, iyo qPCR qaybaha si gaar 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 |
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