RTL Reverse Transcriptase
ʻO ka RTL reverse transcriptase he DNA polymerase hilinaʻi RNA template i nele i ka hana exonuclease 3'→5' a he hana RNase H.Hiki i kēia enzyme ke hoʻohana i ka RNA ma ke ʻano he laʻana no ka hoʻohui ʻana i kahi kaula hoʻohui o DNA, hiki ke hoʻohana ʻia i ka synthesis cDNA mua, ʻoi aku no ka RT-LAMP (loop-mediated isothermal amplification).Hoʻohālikelike ʻia me RTL reverse transcriptase 1.0, ʻoi aku ka maikaʻi o ka naʻau, ʻoi aku ka ikaika o ka thermal stability, a ʻoi aku ka paʻa o ka hopena ma 65 ° C.Hiki ke hoʻohana ʻia ka RTL reverse transcriptase (glycerol free) e hoʻomākaukau i nā hoʻomākaukau lyophilized, lyophilized RT-LAMP reagents etc.
Wehewehe Unite
Hoʻokahi pūʻulu hoʻohui 1 nmol o dTTP i loko o nā mea hiki ke hoʻokahe i ka waika i loko o 20 mau minuke ma 50°C me ka hoʻohana ʻana i ka poly(A)•oligo(dT)25 ma ke ʻano he template-primer.
Nā ʻāpana
| ʻāpana | 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 |
Kūlana Waihona
ʻO ka halihali ma lalo o 0°C a mālama ʻia ma -25°C~-15°C.
Ka Mana Mana
- Koena Hana oEndonuclease:ʻO ka hopena 50 μL i loaʻa ka 1 μg o λDNA a me 15 mau ʻāpana o RTL2.0 incubated no 16 mau hola ma 37 ℃ e hōʻike ana i ke ʻano like me ka mana maikaʻi ʻole e ka electrophoresis gel.
- Koena Hana oExonuclease:ʻO ka hopena 50 μL i loaʻa ka 1 μg o Hind Ⅲ digested λDNA a me 15 mau ʻāpana o RTL2.0 incubated no 16 mau hola ma 37 ℃ e hōʻike ana i ke ʻano like me ka mana maikaʻi ʻole e ka electrophoresis gel.
- Koena Hana oNickase:ʻO kahi hopena 50 μL i loaʻa ka 1 μg o supercoiled pBR322 a me 15 mau ʻāpana o RTL2.0 incubated no 4 mau hola ma 37 ° C e hōʻike ana i ke ʻano like me ka mana maikaʻi ʻole e ka electrophoresis gel.
- Koena Hana oRNase:ʻO ka hopena 10 μL i loaʻa ka 0.48 μg o MS2 RNA a me 15 mau ʻāpana o RTL2.0 incubated no 4 mau hola ma 37 ° C e hōʻike ana i ke ʻano like me ka mana maikaʻi ʻole e ka electrophoresis gel.
- E. coli gDNA:Ana meE.coliʻO nā pahu ʻike HCD kikoʻī ， 15 mau ʻāpana o RTL2.0 aia ma lalo o 1E. coligenome.
Hoʻonohonoho ʻana
cDNA Synthesis Protocol
| Nā ʻāpana | Volume |
| Palapala RNA a | koho |
| Oligo(dT) 18~25(50uM) a i ʻole ka hui ʻana o ke kumu mua (60uM) | 2 μL |
| Hui dNTP (10mM kēlā me kēia) | 1 μL |
| ʻO ka mea pale RNase (40U/uL) | 0.5 μL |
| RTL Reverse Transcriptase 2.0 (15U/uL) | 0.5 μL |
| 10×HC RTL Buffer | 2 μL |
| Wai Nuclease-free | A hiki i 20 μL |
Nā memo:
1) ʻO ka nui o ka RNA i ʻōlelo ʻia he 1ng ~ 1μg
2) ʻO 50ng ~ 100ng ka mea i manaʻo ʻia o ka mRNA
Thermo-paikikala Nā kūlana no ka hana maʻamau pane:
| Mahana (°C) | Manawa |
| 25 °Ca | 5 min |
| 55 °C | 10 minb |
| 80 °C | 10 min |
Nā memo:
1) Inā hoʻohana ʻia ʻo Random Primer Mix, kahi hana incubation ma 25 ° C.
2) Inā hoʻohana ʻia ka hui mua ʻana, kahi incubation step ma 55 ° C no 10 ~ 30mins.
RT-LAMP Protocol
| Nā ʻāpana | Volume | Hoʻopaʻa hope |
| Kāpena RNA | koho | ≥10 kope |
| Hui dNTP (10mM) | 3.5 μL | 1.4 mM |
| FIP/BIP Nā Papahana (25×) | 1 μL | 1.6 μM |
| F3/B3 Puhi (25×) | 1 μL | 0.2 μM |
| Nā Papa LoopF/LoopB (25×) | 1 μL | 0.4 μM |
| ʻO ka mea pale RNase (40U/μL) | 0.5 μL | 20 U/Pane |
| RTL Reverse Transcriptase 2.0 (15U/μL) | 0.5 μL | 7.5 U/Pane |
| Bst V2 DNA Polymerase (8U/μL) | 1 μL | 8 U/Pane |
| MgSO4 (100mM) | 1.5 μL | 6 mM (Ka huina 8 mM) |
| 10×HC RTL Buffer (a i ʻole 10×HC Bst V2 Buffer) | 2.5 μL | 1 × (2mM Mg2+) |
| Wai Nuclease-free | A hiki i ka 25 μL | - |
Nā memo:
1) E hui pū me ka vortexing a me ka centrifuge pōkole e hōʻiliʻili.ʻO ka incubation wela mau ma 65 ° C no 1 hola.
2) Hiki ke hoʻohana ʻia nā mea hoʻopaʻa ʻelua a ua like ka haku mele ʻana.
Nā memo
1. ʻO kēia huahana e hana i kahi paʻa keʻokeʻo ke mālama ʻia ma -20 °C.Wehe i waho mai -20 ° C a kau i ka hau no kahi o 10 mau minuke.Ma hope o ka hoʻoheheʻe ʻana, hiki ke hoʻohana ʻia ma ka lulu ʻana a me ka hui ʻana.
2. Hiki ke mālama ʻia ka huahana cDNA ma -20°C a i ʻole -80°C a hoʻohana koke ʻia no ka hopena PCR.
3. No ka pale ʻana i ka haumia RNase, e ʻoluʻolu e mālama i ka maʻemaʻe o ka wahi hoʻokolohua, a hoʻokomo i nā mīkina lima maʻemaʻe a me nā masks i ka wā o ka hana.
