I-RTL Reverse Transcriptase
I-RTL reverse transcriptase iyi-DNA polymerase encike kusifanekiso se-RNA engenawo umsebenzi we-exonuclease ongu-3'→5' futhi inomsebenzi we-RNase H.Le enzyme ingasebenzisa i-RNA njengesifanekiso ukuze ihlanganise umucu ohambisanayo we-DNA, ongasetshenziswa ekuhlanganiseni kwe-cDNA yomucu wokuqala, ikakhulukazi i-RT-LAMP (i-loop-mediated isothermal amplification).Uma kuqhathaniswa ne-RTL reverse transcriptase 1.0, ukuzwela kuthuthukiswa kakhulu, ukuzinza kwe-thermal kunamandla, futhi ukusabela ku-65°C kuzinze kakhudlwana.I-RTL reverse transcriptase (i-glycerol free) ingasetshenziswa ukulungisa amalungiselelo e-lyophilized, ama-reagents e-RT-LAMP a-lyophilized njll.
Incazelo Yeyunithi
Iyunithi eyodwa ihlanganisa i-nmol engu-1 ye-dTTP ezintweni ezine-asidi eningi emizuzwini engu-20 ku-50°C isebenzisa i-poly(A)•oligo(dT)25 njenge-template-primer.
Izingxenye
| Isakhi | HC5008A-01 | HC5008A-02 | HC5008A-03 |
| I-RTL Reverse Transcriptase (I-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 |
| I-MgSO4 (100mM) | 1.5 mL | 2×1.5 mL | 3 × 10 mL |
Isimo Sesitoreji
Ukuthutha ngaphansi kuka-0°C futhi kugcinwe ku -25°C~-15°C.
Ikhwalithi yokulawula
- Umsebenzi Osele weEi-nuclease:Ukusabela okungu-50 μL okuqukethe u-1 μg we-λDNA namayunithi angu-15 e-RTL2.0 efukanyelwe amahora angu-16 ngo-37 ℃ kubonisa iphethini efanayo nokulawula okungalungile ngejeli electrophoresis.
- Umsebenzi Osele weI-Exonuclease:Ukusabela okungu-50 μL okuqukethe u-1 μg we-Hind Ⅲ egayiwe i-λDNA namayunithi angu-15 e-RTL2.0 efukanyelwe amahora angu-16 ngo-37 ℃ ibonisa iphethini efanayo njengokulawula okungekuhle ngejeli electrophoresis.
- Umsebenzi Osele weI-Nickase:Ukusabela okungu-50 μL okuqukethe u-1 μg we-pBR322 ekhohliwe kakhulu namayunithi angu-15 e-RTL2.0 efukanyelwe amahora angu-4 ku-37°C kubonisa iphethini efanayo njengokulawula okungekuhle ngejeli electrophoresis.
- Umsebenzi Osele weRNase:Ukusabela okungu-10 μL okuqukethe u-0.48 μg we-MS2 RNA namayunithi angu-15 e-RTL2.0 efukanyelwe amahora angu-4 ku-37°C kubonisa iphethini efanayo njengokulawula okungekuhle ngejeli electrophoresis.
- E. coli gI-DNA:Kukalwe ngeE.coliamakhithi okuthola i-HCD athile, amayunithi ayi-15 we-RTL2.0 aqukethe ngaphansi kuka-1E. colii-genome.
Ukusethwa kokusabela
I-cDNA Synthesis Protocol
| Izingxenye | Ivolumu |
| Ithempulethi ye-RNA a | ngokuzikhethela |
| I-Oligo(dT) 18~25(50uM) noma i-Random Primer mix(60uM) | 2 ml |
| I-dNTP Mix (10mM ngayinye) | 1 μl |
| I-RNase Inhibitor (40U/uL) | 0.5 μL |
| I-RTL Reverse Transcriptase 2.0 (15U/uL) | 0.5 μL |
| 10×HC RTL Buffer | 2 ml |
| Amanzi Angenayo I-nuclease | Kufika ku-20 μL |
Amanothi:
1) Umthamo onconyiwe weTotal RNA ngu-1ng~1μg
2) Umthamo onconyiwe we-mRNA wawungu-50ng ~ 100ng
Thermo-ukuhamba ngebhayisikili Imibandela yesimiso ukusabela:
| Izinga lokushisa (°C) | Isikhathi |
| 25 °Ca | 5 imiz |
| 55 °C | 10 imizb |
| 80 °C | 10 imiz |
Amanothi:
1) Uma kusetshenziswa i-Random Primer Mix, isinyathelo sokufukamela esingu-25°C.
2) Uma kusetshenziswa ingxube ye-primer eqondiwe, isinyathelo sokufukamela esingu-55°C imizuzu eyi-10~30.
Iphrothokholi ye-RT-LAMP
| Izingxenye | Ivolumu | Ukugxila Kokugcina |
| I-RNA yesifanekiso | ngokuzikhethela | ≥10 amakhophi |
| I-dNTP Mix (10mM) | 3.5 μL | 1.4 mM |
| I-FIP/BIP Primes (25×) | 1 μl | 1.6 μM |
| I-F3/B3 Primes (25×) | 1 μl | 0.2 μM |
| I-LoopF/LoopB Primes (25×) | 1 μl | 0.4 μM |
| I-RNase Inhibitor (40U/μL) | 0.5 μL | 20 U/Ukusabela |
| I-RTL Reverse Transcriptase 2.0 (15U/μL) | 0.5 μL | 7.5 U/Ukusabela |
| I-Bst V2 DNA Polymerase (8U/μL) | 1 μl | 8 U/Ukusabela |
| I-MgSO4 (100mM) | 1.5 μL | 6 mM (Isamba esingu-8 mM) |
| 10×HC RTL Buffer (noma 10×HC Bst V2 Buffer) | 2.5 μL | 1 × (2mM Mg2+) |
| Amanzi Angenayo I-nuclease | Kufika ku-25 μL | - |
Amanothi:
1) Hlanganisa nge-vortexing kanye ne-centrifuge kafushane ukuqoqa.Ukushisa okungaguquki ku-incubation ku-65°C ihora elingu-1.
2) Amabhafa amabili ayasebenzisana futhi anokwakheka okufanayo.
Amanothi
1.Lo mkhiqizo uzokwakha okuqinile okumhlophe uma ugcinwe ku -20 °C.Yikhiphe ku-20°C bese uyibeka eqhweni cishe imizuzu eyi-10.Ngemuva kokuncibilika, ingasetshenziswa ngokuthuthumela nokuxuba.
2.Umkhiqizo we-cDNA ungagcinwa ku -20°C noma -80°C noma usetshenziselwe ukusabela kwe-PCR ngokushesha.
3. Ukuze uvimbele ukungcoliswa kwe-RNase, sicela ugcine indawo yokuhlola ihlanzekile, futhi ugqoke amagilavu namamaski ahlanzekile ngesikhathi sokusebenza.
