RTL Reverse Transcriptase
RTL transcriptase-ka-soo-noqoshada waa RNA-ku-tiirsanaan DNA polymerase ah oo ka maqan 3'→5' dhaqdhaqaaqa exonuclease oo leh waxqabadka RNase H.Insaymiskani waxa uu u isticmaali karaa RNA qaab template ah si uu u soo saaro xadhig dhammaystiran oo DNA ah, kaas oo lagu dabaqi karo isku-xidhka cDNA-xadhka kowaad, gaar ahaan RT-LAMP (loop-mediated isothermal amplification).Marka la barbar dhigo RTL transcriptase 1.0, dareenka si weyn ayaa loo wanaajiyey, xasilloonida kulaylku waa ka xoog badan tahay, falcelinta 65°Cna waa mid deggan.RTL reverse transcriptase (glycerol free) ayaa loo isticmaali karaa si loogu diyaariyo diyaarinta lyophilized, lyophilized RT-LAMP reagents iwm.
Qeexitaan Unug
Hal unug waxa uu ku daraa 1 nmol oo dTTP ah walxo aashito ah 20 daqiiqo gudahood 50°C iyada oo la isticmaalayo poly(A)•oligo(dT)25 as template-primer.
Qaybaha
| Qayb | HC5008A-01 | HC5008A-02 | HC5008A-03 |
| RTL Reverse Transcriptase (Glycerol-free) (15U/μL) | 0.1 ml | 1 ml | 10 ml |
| 10×HC RTL Buffer | 1.5 ml | 4×1.5 ml | 5×10 ml |
| MgSO4 (100mM) | 1.5 ml | 2×1.5 ml | 3×10 ml |
Xaalada Kaydinta
Gaadiidka ka hooseeya 0°C waxaana lagu kaydiyaa -25°C~-15°C.
Xakamaynta tayada
- Hadhaaga HawlahaEndonuclease:Dareen-celinta 50 μL oo ka kooban 1 μg oo λDNA ah iyo 15 unug oo RTL2.0 ah oo lagu shubay 16 saacadood at 37 ℃ waxay muujinaysaa qaab la mid ah sida xakamaynta taban ee jel electrophoresis.
- Hadhaaga HawlahaExonuclease:Falcelinta 50 μL oo ka kooban 1 μg Hind ah Ⅲ dheefshiidka λDNA iyo 15 unug oo RTL2.0 ah oo lagu shubay 16 saacadood at 37 ℃ waxay muujinaysaa qaab la mid ah sida xakamaynta taban ee jel electrophoresis.
- Hadhaaga HawlahaNickase:Falcelinta 50 μL oo ka kooban 1 μg oo ah pBR322 supercoiled iyo 15 unug RTL2.0 ah oo lagu shubay 4 saacadood at 37°C waxay muujinaysaa qaab la mid ah sida xakamaynta taban ee jel electrophoresis.
- Hadhaaga HawlahaRNase:Falcelinta 10 μL oo ka kooban 0.48 μg ee MS2 RNA iyo 15 unug oo RTL2.0 ah oo lagu shubay 4 saacadood oo ah 37°C waxay muujinaysaa qaab la mid ah xakamaynta taban ee jel electrophoresis.
- E. koli gDNA:Lagu cabirayE.coliXirmooyinka ogaanshaha gaarka ah ee HCD, 15 cutub oo RTL2.0 ah ayaa ka kooban wax ka yar 1E. koligenome.
Dejinta falcelinta
CDNA Synthesis Protocol
| Qaybaha | Mugga |
| Template RNA a | ikhtiyaari ah |
| Oligo(dT) 18 ~ 25(50uM) ama Random Primer mix(60uM) | 2 μL |
| Isku-dhafka dNTP (10mM midkiiba) | 1 μL |
| Horjoogaha RNase (40U/ul) | 0.5 μl |
| RTL Reverse Transcriptase 2.0 (15U/ul) | 0.5 μl |
| 10×HC RTL Buffer | 2 μL |
| Biyo aan Nukliyeer lahayn | Ilaa 20 μl |
Xusuusin:
1) Qiyaasta lagu taliyey ee Wadarta RNA waa 1ng ~ 1μg
2) Qiyaasta lagu taliyey ee mRNA waxay ahayd 50ng ~ 100ng
Thermo-Shuruudaha baaskiilka ee joogtada ah falcelin:
| Heerkulka (°C) | Waqtiga |
| 25 °Ca | 5 daqiiqo |
| 55 °C | 10 daqiiqob |
| 80 °C | 10 daqiiqo |
Xusuusin:
1) Haddii Random Primer Mix la isticmaalo, tallabo soo laabasho ah oo ah 25°C.
2) Haddii la isticmaalo isku darka asaasiga ah ee bartilmaameedka ah, tillaabada soo-jiidashada ee 55 ° C 10 ~ 30mins.
Protocol-ka RT-LAMP
| Qaybaha | Mugga | U-fiirsashada kama dambaysta ah |
| Template RNA | ikhtiyaari ah | ≥10 koobi |
| Isku-dhafka dNTP (10mM) | 3.5 μl | 1.4 mm |
| Qorayaasha FIP/BIP (25×) | 1 μL | 1.6 μM |
| Furayaasha F3/B3 (25×) | 1 μL | 0.2 μM |
| LoopF/LoopB Horyaallada (25×) | 1 μL | 0.4 μM |
| Horjoogaha RNase (40U/μL) | 0.5 μl | 20 U / Falcelin |
| RTL Reverse Transcriptase 2.0 (15U/μL) | 0.5 μl | 7.5 U / Falcelinta |
| Bst V2 DNA Polymerase (8U/μL) | 1 μL | 8 U/falcelinta |
| MgSO4 (100mM) | 1.5 μl | 6 mm (Wadarta 8 mM) |
| 10×HC RTL Buffer (ama 10×HC Bst V2 Buffer) | 2.5 μL | 1 × (2mM Mg2+) |
| Biyo aan Nukliyeer lahayn | Ilaa 25 μl | - |
Xusuusin:
1) Isku qas adigoo vortexing iyo centrifuge kooban si aad u ururiso.Kuleylinta heerkulka joogtada ah ee 65 ° C ilaa 1 saac.
2) Labada baffer waa kuwo is dhexgal ah oo isku mid ah.
Xusuusin
1. Alaabtaani waxay sameyn doontaa adag oo cad marka lagu kaydiyo -20 °C.Ka soo saar -20 ° C oo ku rid baraf ilaa 10 daqiiqo.Ka dib dhalaalka, waxaa loo isticmaali karaa ruxin iyo isku darka.
2. Alaabta cDNA waxa lagu kaydin karaa -20°C ama -80°C ama waxa loo isticmaali karaa isla markiiba falcelinta PCR.
3.Si looga hortago faddaraynta RNase, fadlan ka ilaali goobta tijaabada ah nadiif, xidho galoofyada iyo maaskaro nadiif ah inta lagu jiro hawlgalka.
