RTL Reverse Transcriptase
I-RTL reverse transcriptase yi-RNA template exhomekeke kwi-DNA polymerase engenawo 3'→5' umsebenzi we-exonuclease kwaye inomsebenzi we-RNase H.Le enzyme inokusebenzisa i-RNA njenge template ukwenza i-strand ehambelanayo ye-DNA, enokuthi isetyenziswe kwi-first-strand cDNA synthesis, ingakumbi i-RT-LAMP (i-loop-mediated isothermal amplification).Xa kuthelekiswa ne-RTL reverse transcriptase 1.0, uvakalelo luphuculwe kakhulu, ukuzinza kwe-thermal kunamandla, kwaye ukusabela kwi-65 ° C kuzinzile.RTL reverse transcriptase (glycerol free) ingasetyenziselwa ukulungiselela amalungiselelo lyophilized, lyophilized RT-LAMP reagents njl.
Inkcazelo yeyunithi
Iyunithi enye idibanisa i-1 nmol ye-dTTP kwizinto ezine-asidi-precipitable kwimizuzu engama-20 ku-50°C isebenzisa i-poly(A)•oligo(dT)25 njenge-template-primer.
Amacandelo
| Icandelo | HC5008A-01 | HC5008A-02 | HC5008A-03 |
| I-RTL Reverse Transcriptase (iGlycerol-free) (15U/μL) | 0.1 ml | 1 ml | 10 ml |
| 10×HC RTL Buffer | 1.5 ml | 4×1.5 mL | 5×10 mL |
| I-MgSO4 (100mM) | 1.5 ml | 2×1.5 mL | 3×10 mL |
Imeko yoGcino
Ukuthutha ngaphantsi kwe-0°C kwaye igcinwe kwi-25°C~-15°C.
Ulawulo lwemeko
- Umsebenzi oshiyekileyo weEndonuclease:I-50 μL yokusabela equlethe i-1 μg ye-λDNA kunye neeyunithi ezili-15 ze-RTL2.0 ezifukanyelwe iiyure eziyi-16 kwi-37 ℃ ibonisa ipateni efanayo njengolawulo olubi nge-gel electrophoresis.
- Umsebenzi oshiyekileyo weI-Exonuclease:Ukusabela kwe-50 μL equlethe i-1 μg ye-Hind Ⅲ yetyiswe i-λDNA kunye neeyunithi ezili-15 ze-RTL2.0 ezifukanywe iiyure ezili-16 kuma-37 ℃ zibonisa ipateni efanayo njengolawulo olubi ngejeli ye-electrophoresis.
- Umsebenzi oshiyekileyo weI-Nickase:Ukusabela kwe-50 μL equlethe i-1 μg ye-pBR322 ene-supercoiled kunye neeyunithi ezili-15 ze-RTL2.0 ezifukanyelwe iiyure ezi-4 kwi-37 ° C zibonisa ipateni efanayo njengolawulo olubi nge-gel electrophoresis.
- Umsebenzi oshiyekileyo weRNase:Ukusabela kwe-10 μL equlethe i-0.48 μg ye-MS2 RNA kunye neeyunithi ezili-15 ze-RTL2.0 ezifukanyelwe iiyure ezi-4 kwi-37 ° C zibonisa ipateni efanayo njengolawulo olubi nge-gel electrophoresis.
- E. coli gI-DNA:Kulinganiswa ngeE.coliiikhithi ezithile zokufumanisa i-HCD, iiyunithi ezili-15 ze-RTL2.0 iqulethe ngaphantsi kwe-1E. coliigenome.
Ukuseta iRection
iProtokholi yoHlanganiso lwe-cDNA
| Amacandelo | Umthamo |
| Isakhelo se-RNA a | ngokuzikhethela |
| Oligo(dT) 18~25(50uM) okanye Random Primer mix(60uM) | 2 μL |
| I-dNTP Mix (10mM nganye) | 1 μL |
| I-RNase Inhibitor (40U/uL) | 0.5 μL |
| I-RTL Reverse Transcriptase 2.0 (15U/uL) | 0.5 μL |
| 10×HC RTL Buffer | 2 μL |
| Amanzi angenaNyukliya | Ukuya kuthi ga kwi-20 μL |
Amanqaku:
I-1) Umlinganiselo ocetyiswayo we-RNA iyonke yi-1ng ~ 1μg
2) Idosi ecetyiswayo ye-mRNA yayiyi-50ng ~ 100ng
Thermo-ukukhwela ibhayisekile Iimeko zesiqhelo impendulo:
| Ubushushu (°C) | Ixesha |
| 25 °Ca | 5 imiz |
| 55 °C | 10minsb |
| 80 °C | 10mins |
Amanqaku:
1) Ukuba iRandom Primer Mix iyasetyenziswa, inyathelo lokufukamela kuma-25°C.
2) Ukuba i-target primer mix isetyenzisiweyo, inyathelo lokufukamela kwi-55 ° C kwi-10 ~ 30mins.
Inkqubo ye-RT-LAMP
| Amacandelo | Umthamo | Ukugxininiswa kokugqibela |
| Ithempleyithi ye-RNA | ngokuzikhethela | ≥10 iikopi |
| Umxube we-dNTP (10mM) | 3.5 μL | 1.4 mm |
| FIP/BIP Primers (25×) | 1 μL | 1.6 μM |
| F3/B3 IiPrimers (25×) | 1 μL | 0.2 μM |
| I-LoopF/LoopB Primers (25×) | 1 μL | 0.4 μM |
| I-RNase Inhibitor (40U/μL) | 0.5 μL | 20 U/Reaction |
| I-RTL Reverse Transcriptase 2.0 (15U/μL) | 0.5 μL | 7.5 U/Ukusabela |
| I-Bst V2 DNA Polymerase (8U/μL) | 1 μL | 8 U/Ukusabela |
| I-MgSO4 (100mM) | 1.5 μL | 6 mM (i-8 mM iyonke) |
| 10×HC RTL Buffer (okanye 10×HC Bst V2 Buffer) | 2.5 μL | 1 × (2mM Mg2+) |
| Amanzi angenaNyukliya | Ukuya kuthi ga kwi-25 μL | - |
Amanqaku:
1) Hlanganisa nge-vortexing kunye ne-centrifuge ngokufutshane ukuqokelela.Ukufudumala rhoqo kwi-65 ° C ngeyure enye.
2) Ezi zithinteli zimbini ziyasebenzisana kwaye zinesakhiwo esifanayo.
Amanqaku
1.Le mveliso iyakwenza okuqinileyo okumhlophe xa igcinwe kwi -20 °C.Yikhuphe kwi-20 ° C kwaye uyibeke emkhenkceni malunga nemizuzu eyi-10.Emva kokunyibilika, ingasetyenziselwa ukugubha kunye nokuxuba.
2.Imveliso ye-cDNA inokugcinwa ku -20°C okanye -80°C okanye isetyenziswe ngoko nangoko kwi-PCR reaction.
3.Ukuthintela ukungcoliseka kwe-RNase, nceda ugcine indawo yovavanyo icocekile, kwaye unxibe iiglavu ezicocekileyo kunye nemaski ngexesha lokusebenza.
