M-MLV Reverse Transcriptase
RevScript Reverse transcriptase waxaa lagu helaa farsamada injineernimada hidaha.Waxay leedahay awoodda isku-dhafka cDNA sare, xasilloonida kulaylka iyo xadka heerkulka falcelinta (ilaa 60°C).Alaabada cDNA-da la farsameeyay waa ilaa 10 kb.Waxay wanaajisaa xidhiidhka qaab-dhismeedka waxayna ku habboon tahay beddelka qoraalka RNA qaab dhismeed sare oo adag ama hiddo-wadaha koobiga hooseeya.
Qaybaha
| Qayb | HC2003B (10,000U) | HC2003B (5*10,000U) | HC2003B (200,000U) |
| RevScript Transcriptase Reverse (200U/μL) | 50 μL | 5×50 μL | 1 ml |
| 5 × RevScript Buffer | 250 μL | 1.25 ml | 5 ml |
Xaalada Kaydinta
Alaabtan waa in lagu kaydiyaa heerkulka -25°C~-15°C muddo 2 sano ah.
Qeexitaan Unug
Hal unug waxa uu ku daraa 1 nmol oo dTTP ah walxo aashito ah oo aan milmi karin 10 daqiiqo gudahood 37°C iyada oo la isticmaalayo Oligo(dT) asal ahaan.
Dejinta falcelinta
1.Denaturation of template RNA (tallaabadani waa ikhtiyaari, denaturation of template RNA caawisaa in la furo qaab-dhismeedka sare, kaas oo hagaajin doona dhalidda ee strand cDNA ugu horeysay.)
| Qaybaha | Mugga (μL) |
| RNase bilaash ah ddH2O | Ilaa 13 |
| Oligo(dT)18 (50 μmol/L) ama Random Primer (50 μmol/L) Ama Farsamooyinka Gaarka ah ee Hiddaha (2 μmol/L) | 1 |
| ama 1 | |
| ama 1 | |
| Habka RNA | X a |
Xusuusin:
1) a: Wadarta RNA: 1-5 ug ama mRNA: 1-500 ng
2) Ku dhaji heerkulka 65 ° C 5 daqiiqo, ka dibna ku wareejin baraf isla markiiba si aad u qaboojiso 2 daqiiqo.Centrifugation kooban si aad u ururiso dareeraha falcelinta, ku dar xalka falcelinta qoraalka gadaasha sida ku cad shaxda soo socota.Si tartiib ah pipette loogu qaso.
1.Diyaarinta isku dhafka falcelinta (mugga 20 μL)
| Qaybaha | Mugga (μL) |
| Isku dhafka tallaabadii hore | 13 |
| 5× kaydka | 4 |
| Isku-dhafka dNTP (10nmol/L) | 1 |
| Gadaal qoraal ah (200 U/μL) | 1 |
| Xakameeyaha RNase (40 U/μL) | 1 |
1.Samee falcelinta xaaladaha soo socda:
| Heerkulka (°C) | Waqtiga |
| 25 °Ca | 5 daqiiqo |
| 42 °Cb | 15-30 daqiiqo |
| 85 °Cc | 5 daqiiqo |
Xusuusin:
1) ah.Ku-soo-baxa heerkulka 25 ° C 5mins ayaa loo baahan yahay oo kaliya isticmaalka hexamers-ka random.Fadlan ka bood tillaabadan markaad isticmaalayso Oligo (dT)18ama Gene Specific Primer.
2) b.Heerkulka dib u celinta lagu taliyey waa 42°C, Qaababka leh qaabdhismeedka sare ee adag ama ka kooban GC sare, waxaa lagu talinayaa in kor loo qaado heerkulka falcelinta ilaa 50-55°C.
3) c.Kuleylinta at 85°C 5mins si loo damiyo qoraalka qoraalka.
4) Alaabta waxaa si toos ah loogu isticmaali karaa falcelinta PCR ama qPCR, ama lagu kaydin karaa -20°C kaydinta muddada gaaban.Waxaa lagu talinayaa in la beddelo alaabta oo lagu kaydiyo at -80 ° C kaydinta muddada-dheer.Iska ilaali baraf-dhalaalid oo joogto ah.
5) Alaabtu waxay ku habboon tahay hal-tallaabo RT-qPCR, waxaa lagu talinayaa in lagu daro 10-20 U dib-u-celinta nidaamka falcelinta 25μL kasta, ama si tartiib tartiib ah u kordhiso qaddarka qoraalka qoraalka ah iyadoo loo eegayo xaaladda dhabta ah.
Xusuusin
1.Fadlan nadiif ka dhig aagga tijaabada;Gacmo-gashiyada nadiifka ah iyo maaskaro waa in la gashado inta lagu jiro hawlgalka.Dhammaan alaabooyinka loo isticmaalo tijaabada waa inay xor u ahaadaan RNase si looga hortago sumowga RNase.
2.Dhammaan nidaamyada waa in lagu sameeyaa baraf si looga hortago hoos u dhaca RNA.
3.Tijaabooyin RNA oo tayo sare leh ayaa lagula talinayaa si loo hubiyo waxtarka sare ee qoraalka qoraalka.
4.Alaabtan waxaa loogu talagalay isticmaalka cilmi-baarista oo keliya.
5.Fadlan ku shaqee jaakadaha shaybaadhka iyo galoofyada la tuuri karo, badbaadadaada awgeed.
