RTL Whakamuri Transcriptase
Ko te RTL whakamuri transcriptase he DNA polymerase e whakawhirinaki ana ki te tauira RNA kaore he mahi 3'→5' exonuclease me te mahi RNase H.Ka taea e tenei enzyme te whakamahi i te RNA hei tauira ki te whakahiato i tetahi whenu taapiri o te DNA, ka taea te tono ki te whenu tuatahi cDNA synthesis, ina koa mo te RT-LAMP (kopiko-mediated isothermal amplification).Ka whakatauritea ki te RTL reverse transcriptase 1.0, ka tino pai ake te maaramatanga, ka kaha ake te pumau o te waiariki, me te tauhohenga i te 65 ° C ka kaha ake.Ka taea te whakamahi RTL whakamuri transcriptase (glycerol free) ki te whakarite i nga whakaritenga lyophilized, lyophilized RT-LAMP reagents etc.
Whakamaramatanga Waeine
Kotahi te waeine e whakauru ana i te 1 nmol o te dTTP ki roto i te waikawa-precipitable rawa i roto i te 20 meneti i te 50°C ma te whakamahi i te poly(A)•oligo(dT)25 hei tauira-primer.
Waehanga
| Waehanga | HC5008A-01 | HC5008A-02 | HC5008A-03 |
| RTL Reverse Transcriptase (Glycerol-free) (15U/μL) | 0.1 mL | 1 mL | 10 mL |
| 10×HC RTL Kaipupuri | 1.5 mL | 4×1.5 mL | 5×10 mL |
| MgSO4 (100mM) | 1.5 mL | 2×1.5 mL | 3×10 mL |
Tikanga Rokiroki
Te kawe i raro i te 0°C ka penapena ki te -25°C~-15°C.
Mana Kounga
- Mahi Toenga oEndonuclease:Ko te tauhohenga 50 μL kei roto te 1 μg o te λDNA me te 15 waeine o RTL2.0 i whakakororiahia mo te 16 haora i te 37 ℃ e whakaatu ana i te tauira rite ki te mana kino e te electrophoresis gel.
- Mahi Toenga oExonuclease:Ko te tauhohenga 50 μL kei roto te 1 μg o Hind Ⅲ te λDNA kua keri me te 15 waeine o RTL2.0 i whakakororia mo te 16 haora i te 37 ℃ e whakaatu ana i te tauira rite ki te mana kino e te electrophoresis gel.
- Mahi Toenga oNickase:He 50 μL tauhohenga kei roto 1 μg o supercoiled pBR322 me 15 waeine o RTL2.0 incubated mo 4 haora i 37°C whakaatu rite tauira rite te mana kino i te electrophoresis gel.
- Mahi Toenga oRNase:He 10 μL tauhohenga kei roto 0.48 μg o MS2 RNA me 15 waeine o RTL2.0 incubated mō te 4 haora i te 37°C whakaatu rite tauira rite mana kino i te electrophoresis gel.
- E. coli gDNA:Ine ki teE.coliKo nga kete rapunga HCD motuhake，15 nga waeine o RTL2.0 kei raro iho i te 1E. coligenome.
Tauhohenga Tatūnga
Kawa Synthesis cDNA
| Waehanga | Rōrahi |
| Tauira RNA a | kōwhiringa |
| Oligo(dT) 18~25(50uM) Rahui Taketake ranei (60uM) | 2 μL |
| Whakaranu dNTP (10mM ia) | 1 μL |
| Rnaase Inhibitor (40U/uL) | 0.5 μL |
| RTL Whakamuri Transcriptase 2.0 (15U/uL) | 0.5 μL |
| 10×HC RTL Kaipupuri | 2 μL |
| Wai kore-nuclease | Tae atu ki te 20 μL |
Tuhipoka:
1) Ko te inenga tūtohutia o te Tapeke RNA ko 1ng~1μg
2) Ko te inenga tūtohutia o te mRNA ko 50ng~100ng
Thermo-paihikara Nga tikanga mo te mahinga tauhohenga:
| Paemahana (°C) | Te wa |
| 25 °Ca | 5min |
| 55 °C | 10 menetib |
| 80 °C | 10 meneti |
Tuhipoka:
1) Mēnā ka whakamahia te Random Primer Mix, he taahiraa heki i te 25°C.
2) Mēnā ka whakamahia te ranunga tuatahi, he taahiraa heki i te 55°C mo te 10~30mins.
Kawa RT-LAMP
| Waehanga | Rōrahi | Te Whakamutunga Whakamutunga |
| RNA tauira | kōwhiringa | ≥10 kape |
| Whakaranu dNTP (10mM) | 3.5 μL | 1.4 mM |
| FIP/BIP Primer (25×) | 1 μL | 1.6 μM |
| F3/B3 Pūmatua (25×) | 1 μL | 0.2 μM |
| RoopF/LoopB Primer (25×) | 1 μL | 0.4 μM |
| Te aukati RNase (40U/μL) | 0.5 μL | 20 U/Tauhohenga |
| RTL Whakamuri Transcriptase 2.0 (15U/μL) | 0.5 μL | 7.5 U/Tauhohenga |
| Bst V2 DNA Polymerase (8U/μL) | 1 μL | 8 U/Tauhohenga |
| MgSO4 (100mM) | 1.5 μL | 6 mM (Tapeke 8 mM) |
| 10×HC RTL Buffer (10×HC Bst V2 Kaipupuri ranei) | 2.5 μL | 1 × (2mM Mg2+) |
| Wai kore-nuclease | Tae atu ki te 25 μL | - |
Tuhipoka:
1) Whakaranuhia ma te vortexing me te centrifuge poto hei kohi.Te whakamaaramatanga o te pāmahana i te 65°C mo te 1 haora.
2) He mahi tahi nga parepare e rua, he rite tonu te hanganga.
Notes
1.Ko tenei hua ka hanga he totoka ma ina rongoa i te -20 °C.Tangohia mai i te -20°C ka maka ki runga tio mo te 10 meneti pea.I muri i te rewa, ka taea te whakamahi ma te wiri me te whakaranu.
2.Ka taea te rongoa i te hua cDNA ki -20°C, -80°C ranei ka whakamahia tonu mo te tauhohenga PCR.
3. Hei aukati i te poke RNase, kia ma tonu te waahi whakamatautau, ka mau i nga karapu ma me nga kanohi kanohi i te wa e mahi ana.
