RTL Reverse Transcriptase
RTL reverse transcriptase ndi DNA polymerase yodalira template ya RNA yomwe ilibe 3'→ 5' ntchito yotulutsa exonuclease ndipo imakhala ndi zochita za RNase H.Enzyme iyi imatha kugwiritsa ntchito RNA ngati template kuti ipangitse chingwe chothandizira cha DNA, chomwe chitha kugwiritsidwa ntchito popanga chingwe choyamba cha cDNA, makamaka cha RT-LAMP (loop-mediated isothermal amplification).Poyerekeza ndi RTL reverse transcriptase 1.0, kukhudzidwa kumakhala bwino kwambiri, kukhazikika kwa kutentha kumakhala kolimba, ndipo zomwe zimachitika pa 65 ° C zimakhala zokhazikika.RTL reverse transcriptase (glycerol free) angagwiritsidwe ntchito pokonzekera lyophilized kukonzekera, lyophilized RT-LAMP reagents etc.
Tanthauzo la Chigawo
Chigawo chimodzi chimaphatikiza 1 nmol ya dTTP muzinthu zotsika acid mu mphindi 20 pa 50°C pogwiritsa ntchito poly(A)•oligo(dT)25 ngati template-primer.
Zigawo
| Chigawo | Mtengo wa HC5008A-01 | Mtengo wa HC5008A-02 | Mtengo wa HC5008A-03 |
| RTL Reverse Transcriptase (Glycerol-free) (15U/μL) | 0.1 ml pa | 1 ml | 10 ml pa |
| 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 |
Mkhalidwe Wosungira
Kuyenda pansi pa 0 ° C ndikusungidwa pa -25 ° C ~ -15 ° C.
Kuwongolera Kwabwino
- Ntchito Yotsalira yaEndonuclease:Mawonekedwe a 50 μL okhala ndi 1 μg ya λDNA ndi mayunitsi 15 a RTL2.0 olumikizidwa kwa maola 16 pa 37 ℃ amawonetsa njira yofanana ndi kuwongolera koyipa kwa gel electrophoresis.
- Ntchito Yotsalira yaExonuclease:Mawonekedwe a 50 μL okhala ndi 1 μg ya Hind Ⅲ yogayidwa λDNA ndi mayunitsi 15 a RTL2.0 okulungidwa kwa maola 16 pa 37 ℃ akuwonetsa njira yofanana ndi kuwongolera koyipa kwa gel electrophoresis.
- Ntchito Yotsalira yaNickase:50 μL reaction yokhala ndi 1 μg ya supercoiled pBR322 ndi 15 mayunitsi a RTL2.0 incubated kwa 4 hours pa 37°C amasonyeza chitsanzo chimodzimodzi monga kulamulira zoipa ndi gel electrophoresis.
- Ntchito Yotsalira yaRNase:Mayankho a 10 μL okhala ndi 0.48 μg ya MS2 RNA ndi mayunitsi 15 a RTL2.0 okulungidwa kwa maola 4 pa 37°C akuwonetsa njira yofanana ndi kuwongolera koyipa kwa gel electrophoresis.
- E. koli gDNA:Kuyezedwa ndiE.colizida zapadera za HCD, magawo 15 a RTL2.0 ali ndi zosakwana 1E. koligenome.
Kukonzekera kwa Reaction
cDNA Synthesis Protocol
| Zigawo | Voliyumu |
| Chithunzi cha RNA a | kusankha |
| Oligo(dT) 18~25(50uM) or Random Primer mix(60uM) | 2 ml |
| dNTP Mix (10mM iliyonse) | 1 ml |
| RNase Inhibitor (40U/uL) | 0.5 μL |
| RTL Reverse Transcriptase 2.0 (15U/uL) | 0.5 μL |
| 10 × HC RTL Buffer | 2 ml |
| Madzi opanda nyukiliya | Mpaka 20 μL |
Ndemanga:
1) Mlingo woyenera wa Total RNA ndi 1ng ~ 1μg
2) Mlingo wovomerezeka wa mRNA unali 50ng ~ 100ng
Thermo-Pang'onopang'ono Zoyenera kuchita chizolowezi zomwe:
| Kutentha (°C) | Nthawi |
| 25 °Ca | 5 mins |
| 55 °C | 10 minsb |
| 80 °C | 10 mins |
Ndemanga:
1) Ngati Random Primer Mix agwiritsidwa ntchito, sitepe yoyamwitsa pa 25°C.
2) Ngati chandamale chisakanizo chikugwiritsidwa ntchito, sitepe yoyambira pa 55 ° C kwa 10 ~ 30mins.
RT-LAMP Protocol
| Zigawo | Voliyumu | Kukhazikika Kwambiri |
| Chithunzi cha RNA | kusankha | ≥10 makope |
| dNTP Mix (10mM) | 3.5 μl | 1.4 mm |
| FIP/BIP Primers (25×) | 1 ml | 1.6 μM |
| Zoyamba za F3/B3 (25×) | 1 ml | 0.2 μm |
| Zoyamba za LoopF/LoopB (25×) | 1 ml | 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 ml | 8 U/Kuchita |
| MgSO4 (100mM) | 1.5 μL | 6 mM (Zokwanira 8 mM) |
| 10 × HC RTL Buffer (kapena 10 × HC Bst V2 Buffer) | 2.5 μL | 1 × (2mM Mg2+) |
| Madzi opanda nyukiliya | Mpaka 25 μL | - |
Ndemanga:
1) Sakanizani ndi vortexing ndi centrifuge mwachidule kuti mutenge.Kutentha kosasintha kwa 65 ° C kwa ola limodzi.
2) Ma buffers awiriwa ndi ogwirizana ndipo ali ndi mawonekedwe ofanana.
Zolemba
1.Chinthu ichi chipanga cholimba choyera chikasungidwa pa -20 °C.Chotsani pa -20 ° C ndikuyika pa ayezi kwa mphindi 10.Pambuyo pa kusungunuka, ingagwiritsidwe ntchito pogwedeza ndi kusakaniza.
2.The cDNA mankhwala akhoza kusungidwa pa -20°C kapena -80°C kapena kugwiritsidwa ntchito nthawi yomweyo PCR reaction.
3.Kuti mupewe kuipitsidwa kwa RNase, chonde sungani malo oyesera, ndi kuvala magolovesi ndi masks oyera panthawi yogwira ntchito.
