RTL Reverse Transcriptase
RTL reverse transcriptase ke DNA polymerase e itšetlehileng ka thempleite ea RNA e hlokang tšebetso ea 3'→5' exonuclease 'me e na le tšebetso ea RNase H.Enzyme ena e ka sebelisa RNA e le thempleite ho kopanya tlhale e tlatsanang ea DNA, e ka sebelisoang ho motsoako oa pele oa cDNA, haholo-holo bakeng sa RT-LAMP (loop-mediated isothermal amplification).Ha ho bapisoa le RTL reverse transcriptase 1.0, kutloisiso e ntlafatsoa haholo, botsitso ba mocheso bo matla, 'me karabelo ea 65 ° C e tsitsitse haholoanyane.RTL reverse transcriptase (glycerol free) e ka sebelisoa ho lokisa litokisetso tsa lyophilized, li-reagents tsa RT-LAMP tse nang le lyophilized joalo-joalo.
Tlhaloso ea Yuniti
Yuniti e le 'ngoe e kenyelletsa 1 nmol ea dTTP ho lintho tse nang le asiti ka metsotso e 20 ho 50°C e sebelisa poly(A)•oligo(dT)25 e le template-primer.
Likaroloana
| Karolo | HC5008A-01 | HC5008A-02 | HC5008A-03 |
| RTL Reverse Transcriptase (Mahala Glycerol) (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 |
Boemo ba polokelo
Lipalangoang ka tlas'a 0°C 'me li bolokoe ho -25°C~-15°C.
Taolo ea Boleng
- Mosebetsi o setseng oaEkokoana-hloko:Karabelo ya 50 μL e nang le 1 μg ya λDNA le diyuniti tse 15 tsa RTL2.0 tse ekeditsweng dihora tse 16 ho 37 ℃ e bontsha paterone e tshwanang le taolo e mpe ka gel electrophoresis.
- Mosebetsi o setseng oaExonuclease:Maikutlo a 50 μL a nang le 1 μg ea Hind Ⅲ e chekiloeng λDNA le li-unit tse 15 tsa RTL2.0 tse ekelitsoeng lihora tse 16 ho 37 ℃ e bonts'a mokhoa o ts'oanang le taolo e mpe ka gel electrophoresis.
- Mosebetsi o setseng oaNickase:Maikutlo a 50 μL a nang le 1 μg ea supercoiled pBR322 le li-unit tse 15 tsa RTL2.0 tse ekelitsoeng lihora tse 4 ho 37 ° C e bonts'a mokhoa o ts'oanang le taolo e mpe ka gel electrophoresis.
- Mosebetsi o setseng oaRNase:Karabelo ea 10 μL e nang le 0.48 μg ea MS2 RNA le li-unit tse 15 tsa RTL2.0 tse ekelitsoeng lihora tse 4 ho 37 ° C li bonts'a mokhoa o ts'oanang le taolo e mpe ka gel electrophoresis.
- E. coli gDNA:E lekantsoeng kaE.coliLisebelisoa tse khethehileng tsa ho lemoha tsa HCD, likarolo tse 15 tsa RTL2.0 li na le ka tlase ho 1E. coligenome.
Rection Setup
cDNA Synthesis Protocol
| Likaroloana | Bolumo |
| Setšoantšo sa RNA a | boikhethelo |
| Oligo(dT) 18~25(50uM) kapa Motsoako o Random Primer(60uM) | 2 μL |
| dNTP Mix (10mM e 'ngoe le e 'ngoe) | 1 μL |
| RNase Inhibitor (40U/uL) | 0.5 μL |
| RTL Reverse Transcriptase 2.0 (15U/uL) | 0.5 μL |
| 10×HC RTL Buffer | 2 μL |
| Metsi a se nang Nuclease | Ho fihlela ho 20 μL |
Lintlha:
1) Tekanyetso e khothalletsoang ea Total RNA ke 1ng ~ 1μg
2) Tekanyetso e khothaletsoang ea mRNA e ne e le 50ng ~ 100ng
Thermo-cycling Maemo bakeng sa tloaelo karabelo:
| Mocheso (°C) | Nako |
| 25 °Ca | 5 mets |
| 55 °C | 10metsotsob |
| 80 °C | 10metsotso |
Lintlha:
1) Haeba Random Primer Mix e sebelisoa, mohato oa ho incubating ho 25°C.
2) Haeba sepheo sa "primer mix" se sebelisoa, mohato oa ho incubation ho 55 ° C bakeng sa 10 ~ 30mins.
RT-LAMP Protocol
| Likaroloana | Bolumo | Boikutlo ba ho Qetela |
| Setšoantšo sa RNA | boikhethelo | ≥10 likopi |
| dNTP Mix (10mM) | 3.5 μL | 1.4 mM |
| FIP/BIP Primes (25×) | 1 μL | 1.6 μM |
| F3/B3 Primers (25×) | 1 μL | 0.2 μM |
| Li-Prime tsa LoopF/LoopB (25×) | 1 μL | 0.4 μM |
| RNase Inhibitor (40U/μL) | 0.5 μL | 20 U/Karabelo |
| RTL Reverse Transcriptase 2.0 (15U/μL) | 0.5 μL | 7.5 U/Karabelo |
| Bst V2 DNA Polymerase (8U/μL) | 1 μL | 8 U/Karabelo |
| MgSO4 (100mM) | 1.5 μL | 6 mM (Kakaretso 8 mM) |
| 10×HC RTL Buffer (kapa 10×HC Bst V2 Buffer) | 2.5 μL | 1 × (2mM Mg2+) |
| Metsi a se nang Nuclease | Ho fihlela ho 25 μL | - |
Lintlha:
1) Kopanya ka vortexing le centrifuge ka bokhutšoanyane ho bokella.Incubation ea kamehla ea mocheso ho 65°C bakeng sa hora e le 1.
2) Li-buffer tse peli lia sebelisana 'me li na le sebopeho se tšoanang.
Lintlha
1.Sehlahisoa sena se tla theha sepakapaka se tšoeu ha se bolokiloe ho -20 °C.E ntše ho -20 ° C 'me ue behe holim'a leqhoa ka metsotso e ka bang 10.Ka mor'a ho qhibiliha, e ka sebelisoa ka ho sisinyeha le ho kopanya.
2.Sehlahisoa sa cDNA se ka bolokoa ho -20°C kapa -80°C kapa sa sebelisoa hang-hang bakeng sa karabelo ea PCR.
3.Ho thibela tšilafalo ea RNase, ka kopo boloka sebaka sa liteko se hloekile, 'me u roale liatlana le limaske tse hloekileng nakong ea ts'ebetso.
