RTL Inyandikomvugo
RTL revers transcriptase ni RNA yerekana-ishingiye kuri ADN polymerase idafite ibikorwa bya 3 '→ 5' exonuclease kandi ifite ibikorwa bya RNase H.Iyi misemburo irashobora gukoresha RNA nkicyitegererezo cyo guhuza umurongo wuzuzanya wa ADN, ushobora gukoreshwa muburyo bwa cDNA bwa mbere, cyane cyane kuri RT-LAMP (amplification isothermal amplification).Ugereranije na RTL revers transcriptase 1.0, ibyiyumvo byateye imbere cyane, ituze ryumuriro rirakomera, kandi reaction kuri 65 ° C irahagaze neza.RTL revers transcriptase (glycerol yubusa) irashobora gukoreshwa mugutegura imyiteguro ya lyofilize, reofent ya RT-LAMP nibindi.
Igisobanuro
Igice kimwe cyinjiza 1 nmol ya dTTP mubintu byangiza aside muminota 20 kuri 50 ° C ukoresheje poly (A) • oligo (dT) 25 nkicyitegererezo-primer.
Ibigize
| Ibigize | HC5008A-01 | HC5008A-02 | HC5008A-03 |
| RTL Guhindura Inyandiko (Glycerol-yubusa) (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 |
Imiterere y'Ububiko
Ubwikorezi buri munsi ya 0 ° C kandi bubikwa kuri -25 ° C ~ -15 ° C.
Kugenzura ubuziranenge
- Igikorwa gisigaye cyaEndonuclease:50 μL reaction irimo 1 μg ya λDNA hamwe na 15 ya RTL2.0 yashizwemo amasaha 16 kuri 37 ℃ yerekana uburyo bumwe bwo kugenzura nabi na gel electrophorei.
- Igikorwa gisigaye cyaExonuc Please:50 μL reaction irimo 1 μg ya Hind Ⅲ igogorwa λDNA hamwe na 15 ya RTL2.0 yashizwemo amasaha 16 kuri 37 ℃ yerekana uburyo bumwe nubugenzuzi bubi bwa gel electrophorei.
- Igikorwa gisigaye cyaNickase:50 μL reaction irimo 1 μg ya pBR322 ya supercoile hamwe na 15 za RTL2.0 zashyizwe mumasaha 4 kuri 37 ° C yerekana uburyo bumwe no kugenzura nabi na gel electrophorei.
- Igikorwa gisigaye cyaRNase:Imyitwarire ya 10 μL irimo 0.48 μg ya MS2 RNA hamwe na 15 ya RTL2.0 yashizwemo amasaha 4 kuri 37 ° C yerekana uburyo bumwe no kugenzura nabi na gel electrophorei.
- E. coli gADN:Bipimwe hamweE.coliibikoresho byihariye bya HCD byerekana ibikoresho units 15 bya RTL2.0 birimo munsi ya 1E. coligenome.
Gushiraho
cDNA Porotokole
| Ibigize | Umubumbe |
| Inyandikorugero RNA a | bidashoboka |
| Oligo (dT) 18 ~ 25 (50uM) cyangwa Primer Primer ivanze (60uM) | 2 μL |
| DNTP ivanga (10mM buri umwe) | 1 μL |
| Inhibitor ya RNase (40U / uL) | 0.5 μL |
| RTL Guhindura Inyandiko 2.0 (15U / uL) | 0.5 μL |
| 10 × HC RTL Buffer | 2 μL |
| Amazi adafite ingufu | Kugera kuri 20 μL |
Inyandiko:
1) Igipimo gisabwa cya RNA Yuzuye ni 1ng ~ 1μg
2) Igipimo gisabwa cya mRNA cyari 50ng ~ 100ng
Thermo-gusiganwa ku magare Ibintu bisanzwe reaction ：
| Ubushyuhe (° C) | Igihe |
| 25 ° C.a | 5min |
| 55 ° C. | 10minb |
| 80 ° C. | 10min |
Inyandiko:
1) Niba ikoreshwa rya Primer Primer rivanze, intambwe ya incubation kuri 25 ° C.
2) Niba intego ya primer ivanze ikoreshwa, intambwe ya incubation kuri 55 ° C kuri 10 ~ 30mins.
Porotokole ya RT-LAMP
| Ibigize | Umubumbe | Kwibanda kwanyuma |
| Inyandikorugero RNA | bidashoboka | Kopi 10 |
| DNTP ivanga (10mM) | 3.5 μL | 1.4 mM |
| FIP / BIP Primers (25 ×) | 1 μL | 1.6 μM |
| F3 / B3 Primers (25 ×) | 1 μL | 0.2 μM |
| LoopF / LoopB Primers (25 ×) | 1 μL | 0.4 μM |
| Inhibitor ya RNase (40U / μL) | 0.5 μL | 20 U / Igisubizo |
| RTL Guhindura Inyandiko 2.0 (15U / μL) | 0.5 μL | 7.5 U / Igisubizo |
| Bst V2 ADN Polymerase (8U / μL) | 1 μL | 8 U / Igisubizo |
| MgSO4 (100mM) | 1.5 μL | MM 6 (Yose hamwe 8 mM) |
| 10 × Buffer ya HC RTL (cyangwa 10 × HC Bst V2 Buffer) | 2.5 μL | 1 × (2mM Mg2 +) |
| Amazi adafite ingufu | Kugera kuri 25 μL | - |
Inyandiko:
1) Kuvanga na vortexing na centrifuge muri make gukusanya.Ubushyuhe buhoraho kuri 65 ° C kumasaha 1.
2) Buffer ebyiri zirashobora gukorana kandi zifite ibice bimwe.
Inyandiko
1.Ibicuruzwa bizakora ibara ryera iyo bibitswe kuri -20 ° C.Kuramo kuva kuri -20 ° C hanyuma ubishyire kurubura muminota 10.Nyuma yo gushonga, irashobora gukoreshwa mukuzunguza no kuvanga.
2.Ibicuruzwa bya cDNA birashobora kubikwa kuri -20 ° C cyangwa -80 ° C cyangwa bigahita bikoreshwa muburyo bwa PCR.
3.Kwirinda kwanduza RNase, nyamuneka komeza isuku yubushakashatsi, kandi wambare uturindantoki na masike mugihe gikora.
