M-MLV Neoscript Qoraalka Dib u noqoshada
Neoscript Reverse Transcriptase waa qoraal-soo-noqon oo lagu helay baadhista beddelka ee hidda-wadaha M-MLV ee Moloney murine leukemia fayraska asal ahaan iyo muujinta E.coli.Insaymku wuxuu meesha ka saaraa dhaqdhaqaaqa RNase H, wuxuu leeyahay dulqaad heerkul sare ah, wuxuuna ku habboon yahay qoritaanka heerkul sare.Sidaa darteed, waxay waxtar u leedahay baabi'inta saamaynta aan fiicneyn ee qaab-dhismeedka heerka sare ee RNA iyo arrimo aan gaar ahayn oo ku saabsan isku-darka cDNA, waxayna leedahay xasillooni sare iyo dib-u-soo-celinta kartida qoraalka qoraalka.Insaymku waxa uu leeyahay xasillooni sare oo dib u rogida kartida.
Qaybaha
1.200 U/μL Qoraal-qorista Dib-u-Celinta ee Neoscript
2.5 × Xadhig-Xiddiga Koowaad (ikhtiyaar)
* 5 × First-Strand Buffer kuma jiro dNTP, fadlan ku dar dNTP-yada marka la diyaarinayo habka falcelinta
Codsiga lagu taliyay
1.Hal-tallaabo qRT-PCR.
2.ogaanshaha fayraska RNA.
Xaalada Kaydinta
-20°C kaydinta muddada dheer, waa in si fiican loo qaso ka hor inta aan la isticmaalin, iska ilaali baraf-dhalaalid joogto ah.
Qeexitaan Unug
Hal unug waxa uu ku daraa 1 nmol ee dTTP 10 daqiiqo gudahood 37°C iyada oo la isticmaalayo poly(A)•oligo(dT)25sida template/primer.
Xakamaynta tayada
1.SDS-PAGE nadiifinta korantada ee ka weyn 98%.
2.Dareenka weynaynta, xakamaynta dufcaddii, xasilloonida.
3.Wax dhaqdhaqaaq nuclease ah oo dibadda ah ma jiro, ma jiro endonuclease exogenous ama faddarayn exonuuclease
Dejinta Falcelinta ee Xalka Falcelinta Silsiladda Koowaad
1.Diyaarinta isku dhafka falcelinta
| Qaybaha | Mugga |
| Oligo(dT)12-18 Hordhac ama Random Primera Ama Hormoodka Gaarka ah ee Hiddahab | 50 pm |
| 50 pm (20-100 pmol) | |
| 2 pm | |
| 10 mM dNTP | 1 μL |
| Template RNA | Wadarta RNA≤ 5μg;mRNA≤ 1 μg |
| RNase-free dH2O | ilaa 10 μl |
Xusuusin:a/b: Fadlan dooro noocyada kala duwan ee asaasiga ah iyadoo loo eegayo baahiyahaaga tijaabada ah.
2.Kuleyl 65 ° C 5mins oo si degdeg ah ugu qabooji barafka 2 daqiiqo.
3.Ku dar qaybaha soo socda nidaamka kore mugga guud ee 20µL oo si tartiib ah u qas:
| Qaybaha | Mugga (μL) |
| 5 × Xakamaynta-Xiddiga Koowaad | 4 |
| Nuqul ka beddelka Neoscript (200 U/μL) | 1 |
| Xakameeyaha RNase (40 U/μL) | 1 |
| RNase-free dH2O | ilaa 20 μl |
4.Fadlan samee falcelinta iyadoo loo eegayo shuruudaha soo socda:
(1) Haddii Random Primer la isticmaalo, falcelinta waa in lagu sameeyaa 25℃ 10mins, ka dibna 50℃ 30 ~ 60mins;
(2) Haddii Oligo dT ama aasaasiyaal gaar ah la isticmaalo, falcelinta waa in lagu sameeyaa 50 ℃ 30 ~ 60mins.
5.Kuleylka 95℃ 5mins si aad u joojiso Neoscript Reverse Transcriptase oo jooji falcelinta.
6.Alaabooyinka qoraalka dib loo rogay waxaa si toos ah loogu isticmaali karaa falcelinta PCR-ga iyo falcelinta PCR-ga ee fluorescence, ama lagu kaydin karaa -20 ℃ muddo dheer.
PCR Rdhaqdhaqaaqa
1.Diyaarinta isku dhafka falcelinta
| Qaybaha | Xoog-saarid |
| 10 × PCR Buffer (dNTP bilaash ah, Mg²+ bilaash ah) | 1× |
| dNTPs (10mM dNTP kasta) | 200 μM |
| 25 mm MgCl2 | 1-4 mm |
| Taq DNA Polymerase (5U/μL) | 2-2.5 U |
| Hore 1 (10 μM) | 0.2-1 μM |
| Hore 2 (10 μM) | 0.2-1 μM |
| Templatea | ≤10% Xalka Falcelinta Silsiladda Koowaad (2 μL) |
| ddH2O | ilaa 50 μl |
Xusuusin:a: Haddii wax badan lagu daro xalka falcelinta silsiladda koowaad, falcelinta PCR waa la joojin karaa.
2.Habka Falcelinta PCR
| Talaabada | Heerkulka | Waqtiga | Wareegtada |
| Denaturation ka hor | 95℃ | 2-5 daqiiqo | 1 |
| Denaturation | 95℃ | 10-20 ilbiriqsi | 30-40 |
| Annealing | 50-60 ℃ | 10-30 ilbiriqsi | |
| Kordhinta | 72℃ | 10-60 sekan |
Xusuusin
1.Ku habboon hagaajinta heerkulbeegga qoraalka ee u dhexeeya 42 ℃ ~ 55 ℃.
2.Waxay leedahay xasillooni ka wanaagsan, waxay ku habboon tahay heer-kulka sare ee cabbirka qoraalka.Intaa waxaa dheer, waxay ku habboon tahay in si wax ku ool ah looga gudbo gobollada qaab dhismeedka adag ee RNA.Sidoo kale, waxawuxuu ku habboon yahay hal-tilaabo-multixx fluorescence tireed ogaanshaha RT-PCR.
3.Waafaqid wanaagsan ee enzymes-weyniyeedka PCR ee kala duwan waxayna ku habboon tahay dareen-celinta sare ee RT-PCR.
4.Ku habboon dareenka sare hal-tallaabo falcelinta tireed ee Fluorescence ee RT-PCR, si wax ku ool ah u wanaajiso heerka ogaanshaha ee foojignaanta hoose ee jaantusyada.
5.Ku habboon dhismaha maktabadda cDNA.
