RTL Reverse Transcriptase
RTL reverse transcriptase iRNA template-inotsamira DNA polymerase iyo isina 3'→ 5' exonuclease chiitiko uye ine RNase H chiitiko.Iyi enzyme inogona kushandisa RNA setemplate kugadzira tambo inowirirana yeDNA, iyo inogona kuiswa kune yekutanga-strand cDNA synthesis, kunyanya yeRT-LAMP (loop-mediated isothermal amplification).Kuenzaniswa neRTL reverse transcriptase 1.0, kunzwisisika kunovandudzwa zvakanyanya, kugadzikana kwekushisa kwakasimba, uye mhinduro pa 65 ° C inonyanya kugadzikana.RTL reverse transcriptase (glycerol yemahara) inogona kushandiswa kugadzirira lyophilized gadziriro, lyophilized RT-LAMP reagents nezvimwe.
Chikamu Tsanangudzo
Imwe unit inosanganisa 1 nmol ye dTTP muasidhi-inonaya zvinhu mumaminitsi makumi maviri pa50°C ichishandisa poly(A)•oligo(dT)25 setemplate-primer.
Zvikamu
| Chikamu | HC5008A-01 | HC5008A-02 | HC5008A-03 |
| RTL Reverse Transcriptase (Glycerol-isina) (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 |
Storage Condition
Kutakura pasi pe 0°C uye kuchengetwa pa -25°C~-15°C.
Quality Control
- Residual Chiitiko cheEndonuclease:A 50 μL reaction ine 1 μg ye λDNA uye gumi nemashanu mayunitsi eRTL2.0 akavharirwa kwemaawa gumi nematanhatu pa37 ℃ inoratidza maitiro akafanana neakaipa control negel electrophoresis.
- Residual Chiitiko cheExonuclease:A 50 μL reaction ine 1 μg yeHind Ⅲ yakagayiwa λDNA uye gumi nemashanu mayunitsi eRTL2.0 akavharirwa kwemaawa gumi nematanhatu pa37 ℃ anoratidza patani imwechete sekutonga kwakashata negel electrophoresis.
- Residual Chiitiko cheNickase:A 50 μL reaction ine 1 μg ye supercoiled pBR322 uye 15 mayunitsi eRTL2.0 akavharirwa kwemaawa mana pa37°C anoratidza maitiro akafanana neakaipa control negel electrophoresis.
- Residual Chiitiko cheRNase:A 10 μL reaction ine 0.48 μg yeMS2 RNA uye gumi nemashanu mayunitsi eRTL2.0 akavharirwa kwemaawa mana pa37°C anoratidza maitiro akafanana neakaipa control negel electrophoresis.
- E. coli gDNA:Kupimwa neE.colichaiyo HCD yekuona kits, gumi nemashanu zvikamu zveRTL2.0 ine isingasviki 1E. coligenome.
Reaction Setup
cDNA Synthesis Protocol
| Zvikamu | Volume |
| Template RNA a | optional |
| Oligo(dT) 18~25(50uM) kana Random Primer musanganiswa(60uM) | 2 μL |
| dNTP Musanganiswa (10mM imwe neimwe) | 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 |
| Mvura isina Nuclease | Kusvika ku20 μL |
Notes:
1) Muyero wakakurudzirwa weTotal RNA ndeye 1ng ~ 1μg
2) Iyo yakakurudzirwa dosage yemRNA yaive 50ng ~ 100ng
Thermo-bhasikoro Mamiriro ezvinhu emuitiro kuita:
| Tembiricha (°C) | Nguva |
| 25 °Ca | 5 mins |
| 55 °C | 10minsb |
| 80 °C | 10mins |
Notes:
1) Kana Random Primer Mix ikashandiswa, incubation nhanho pa25°C.
2) Kana chinangwa chekutanga musanganiswa chikashandiswa, incubation nhanho pa55 ° C ye10 ~ 30mins.
RT-LAMP Protocol
| Zvikamu | Volume | Final Concentration |
| RNA template | optional | ≥10 makopi |
| dNTP Musanganiswa (10mM) | 3.5 μL | 1.4 mM |
| FIP/BIP Primes (25×) | 1 μL | 1.6 μM |
| F3/B3 Primes (25×) | 1 μL | 0.2 μM |
| LoopF/LoopB Primers (25×) | 1 μL | 0.4 μM |
| RNase Inhibitor (40U/μL) | 0.5 μL | 20 U/Reaction |
| RTL Reverse Transcriptase 2.0 (15U/μL) | 0.5 μL | 7.5 U/Reaction |
| Bst V2 DNA Polymerase (8U/μL) | 1 μL | 8 U/Reaction |
| MgSO4 (100mM) | 1.5 μL | 6 mM (Zvose 8 mM) |
| 10×HC RTL Buffer (kana 10×HC Bst V2 Buffer) | 2.5 μL | 1 × (2mM Mg2+) |
| Mvura isina Nuclease | Kusvika ku25 μL | - |
Notes:
1) Sanganisa nevortexing uye centrifuge muchidimbu kuunganidza.Constant tembiricha incubation pa 65°C kweawa imwe.
2) Iwo maviri mabhafa anodyidzana uye ane akafanana kuumbwa.
Notes
1.Ichi chigadzirwa chichaumba chichena chakasimba kana chakachengetwa pa -20 °C.Bvisa kubva -20 ° C woiisa pachando kwemaminitsi gumi.Mushure mekunyunguduka, inogona kushandiswa nekuzunungusa uye kusanganisa.
2.Chigadzirwa checDNA chinogona kuchengetwa pa -20°C kana -80°C kana kushandiswa nekukasika PCR reaction.
3.Kudzivirira kusvibiswa kweRNase, ndapota chengetedza nzvimbo yekuedza yakachena, uye pfeka magirovhosi akachena uye masikisi panguva yekushanda.
