RTL Reverse Transcriptase
RTL reverse transcriptase bụ DNA polymerase nke dabere na template RNA nke na-enweghị ọrụ exonuclease 3'→5' ma nwee ọrụ RNase H.Enzyme a nwere ike iji RNA dị ka ndebiri iji mepụta eriri DNA na-akwado ya, nke enwere ike itinye ya na njikọ cDNA nke mbụ, ọkachasị maka RT-LAMP (amplification isothermal na-emekọrịta ihe).E jiri ya tụnyere RTL reverse transcriptase 1.0, ezi uche na-eme ka ọ dịkwuo mma, nkwụsi ike na-ekpo ọkụ na-esiwanye ike, na mmeghachi omume na 65 ° C kwụsiri ike.Enwere ike iji RTL reverse transcriptase (glycerol free) kwadebe nkwadebe lyophilized, lyophilized RT-LAMP reagents wdg.
Nkọwa nkeji
Otu nkeji na-etinye 1 nmol nke dTTP n'ime ihe nwere ike ime acid n'ime nkeji 20 na 50°C na-eji poly(A)•oligo(dT)25 dị ka template-primer.
Ngwa
| Akụkụ | HC5008A-01 | HC5008A-02 | HC5008A-03 |
| RTL Reverse Transcriptase (Glycerol n'efu) (15U/μL) | 0,1 ml | 1 ml | 10 ml |
| Ihe nchekwa 10 × HC RTL | 1.5 ml | 4 × 1.5 ml | 5 × 10 ml |
| MgSO4 (100mM) | 1.5 ml | 2 × 1.5 ml | 3 × 10 ml |
Ọnọdụ Nchekwa
Ụgbọ njem n'okpuru 0 Celsius ma debe ya na -25C ~ -15C.
Njikwa ogo
- Residual Ọrụ nkeEndonuclease:Mmeghachi omume 50 μL nwere 1 μg nke λDNA na nkeji 15 nke RTL2.0 etinyere maka awa 16 na 37 ℃ na-egosi otu ụkpụrụ dị ka njikwa na-adịghị mma site na gel electrophoresis.
- Residual Ọrụ nkeExonuclease:Mmeghachi omume 50 μL nwere 1 μg nke Hind Ⅲ gbarie λDNA na nkeji 15 nke RTL2.0 etinyere maka awa 16 na 37 ℃ na-egosi otu ụkpụrụ dị ka njikwa na-adịghị mma site na gel electrophoresis.
- Residual Ọrụ nkeNickase:Mmeghachi omume 50 μL nwere 1 μg nke supercoiled pBR322 na nkeji 15 nke RTL2.0 etinyere maka awa 4 na 37 Celsius C na-egosi otu ụkpụrụ dị ka njikwa na-adịghị mma site na gel electrophoresis.
- Residual Ọrụ nkeRNase:Mmeghachi omume 10 μL nwere 0.48 μg nke MS2 RNA na nkeji 15 nke RTL2.0 etinyere maka awa 4 na 37 Celsius C na-egosi otu ụkpụrụ dị ka njikwa na-adịghị mma site na gel electrophoresis.
- E. coli gDNA:Ejiri ya tụọ yaE.coliNgwa nchọpụta HCD kpọmkwem, nkeji 15 nke RTL2.0 nwere ihe na-erughị 1E. coligenome.
Ntọala mmeghachi omume
cDNA Synthesis Protocol
| Ngwa | Olu |
| Ụdị RNA a | nhọrọ |
| Oligo(dT) 18 ~ 25(50uM) ma ọ bụ Random Primer mix(60uM) | 2 μl |
| Ngwakọta dNTP (10mM nke ọ bụla) | 1 μl |
| Ihe mgbochi RNase (40U/ul) | 0,5 μl |
| RTL ntụgharị ntụgharị 2.0 (15U/ul) | 0,5 μl |
| Ihe nchekwa 10 × HC RTL | 2 μl |
| Mmiri na-enweghị nuklia | Ihe dị ka 20 μl |
ndetu:
1) Usoro akwadoro nke Total RNA bụ 1ng ~ 1μg
2) Usoro akwadoro nke mRNA bụ 50ng ~ 100ng
Thermo-Ịgba ígwè Ọnọdụ maka a eme mmeghachi omume:
| Okpomọkụ (°C) | Oge |
| 25 Celsius Ca | 5mins |
| 55 Celsius C | Nkeji 10b |
| 80 Celsius C | Nkeji 10 |
ndetu:
1) Ọ bụrụ na ejiri Random Primer Mix mee ihe, nzọụkwụ ntinye na 25°C.
2) Ọ bụrụ na a na-eji ngwakọta primer lekwasịrị anya, nzọụkwụ incubation na 55 ° C maka 10 ~ 30mins.
Usoro RT-LAMP
| Ngwa | Olu | Ntụkwasị obi ikpeazụ |
| Ụdị RNA | nhọrọ | ≥10 mbipụta |
| Ngwakọta dNTP (10mM) | 3.5 μl | 1.4 mm |
| FIP/BIP mbụ (25×) | 1 μl | 1.6 μM |
| F3/B3 mbụ (25×) | 1 μl | 0.2 μM |
| LoopF/ LoopB Primers (25×) | 1 μl | 0.4 μM |
| Ihe mgbochi RNase (40U/μL) | 0,5 μl | 20 U/Nmeghachi omume |
| RTL ntụgharị ntụgharị 2.0 (15U/μL) | 0,5 μl | 7.5 U/mmeghachi omume |
| Bst V2 DNA Polymerase (8U/μL) | 1 μl | 8 U/Nmeghachi omume |
| MgSO4 (100mM) | 1.5 μl | 6 mm (Ngụkọta 8 mM) |
| Ihe nchekwa 10×HC RTL (ma ọ bụ 10×HC Bst V2 Buffer) | 2.5 μl | 1 × (2mM mg2+) |
| Mmiri na-enweghị nuklia | Ihe dị ka 25 μl | - |
ndetu:
1) Gwakọta site na vortexing na centrifuge nkenke iji nakọta.Mkpokọta okpomọkụ mgbe niile na 65 Celsius C maka 1 hour.
2) Ihe nchekwa abụọ ahụ na-emekọrịta ihe ma nwee otu ihe mejupụtara.
Ihe ndetu
1. Ngwaahịa a ga-emepụta ihe siri ike na-acha ọcha mgbe echekwara na -20 Celsius.Wepụ ya na -20 Celsius ma tinye ya na ice maka ihe dịka nkeji iri.Mgbe agbazechara, enwere ike iji ya site na ịma jijiji na ịgwakọta.
Enwere ike ịchekwa ngwaahịa cDNA na -20 Celsius ma ọ bụ -80 Celsius ma ọ bụ jiri ya mee ihe ozugbo maka mmeghachi omume PCR.
3.Iji gbochie mmetọ RNase, biko mee ka ebe nnwale ahụ dị ọcha, na-eyikwa uwe na mkpuchi dị ọcha mgbe ị na-arụ ọrụ.
