RTL Reverse Transcriptase
RTL transcriptase contrarium est RNA template-dependens DNA polymerasis quae 3' → 5' exonuclease caret et actio RNase H habet.Hoc enzyme uti potest RNA pro template ad synthesinandam accessionem additivam de DNA, quae applicari potest ad synthesim cDNA primitiva, praesertim pro RT-LAMP (loop-mediatae amplificationis isothermal).Comparato RTL inversa transcriptase 1.0, sensitivum signanter emendatur, scelerisque stabilitas fortior est, et reactio in 65°C stabilior est.RTL transcriptase vicissim (glycerolum liberum) adhiberi possunt ad praeparationes lyophilized praeparandas, reagentes lyophilized RT-LAMP etc.
Unitas Definition
Una unitas 1 nmol de dTTP incorporat in materiam acido-praecipitabilis in 20 minuta ad 50°C utens poly(A) • oligo(dT)25 in primario template.
Components
| Component | HC5008A-01 | HC5008A-02 | HC5008A-03 |
| RTL Reverse Transcriptase (Glycerolum-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 |
Repono Condition
Transportatio sub 0°C et reponenda -25°C~-15°C.
Regimen quālitātis
- RELICTUM activitateEndonuclease:A 50 μL reactionem continens 1 μg de λDNA et 15 unitates RTL2.0 incubatis per 16 horas ante 37 eandem formam ac potestatem negativam per gel electrophoresis ostendit.
- RELICTUM activitateExonuclease:A 50 µL reactionem continens 1 μg Hind Ⅲ digestum λDNA et 15 turmas RTL2.0 incubatas per 16 horas ante 37 eandem formam ac potestatem negativam per electrophoresis gel ostendit.
- RELICTUM activitateNickase:A 50 μL reactionem continens 1 μg supercoilatorum pBR322 et 15 unitates RTL2.0 incubatis per 4 horas ante 37°C eandem formam ac potestatem negativam per electrophoresis gel ostendit.
- RELICTUM activitateRNase:A 10 μL reactionem continens 0.48 μg of MS2 RNA et 15 unitates RTL2.0 incubatis per 4 horas ante 37°C eandem formam ac potestatem negativam ab gel electrophoresi ostendit.
- E. coli gDNA:metiri cumE.colispecifica HCD deprehensio kits XV signa RTL2.0 continet minus quam 1E. coligenome.
Reactionem Setup
cDNA Synthesis Protocol
| Components | Magnitudo |
| Template RNA a | ad libitum |
| Oligo (dT) 18~ 25(50uM) vel Random Primer mix(60uM) | 2 μL |
| dNTP Mix (10mM each) | 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 |
| Nucleas-liberum aquae | Usque ad 20 μL |
Notae:
I) Commendatur dosis totius RNA est 1ng ~ 1μg
II) MRNA suadeo dosis erat 50ng ~ 100ng
Thermo-cycling Conditions in exercitatione reactionem.
| Temperatus (°C) | Tempus |
| 25 °Ca | 5mins |
| 55 °C | 10minsb |
| 80 °C | 10mins |
Notae:
1) Si Random Primer Mix adhibetur, incubationis gradus ad 25°C.
2) Si scopum primario mix adhibetur, incubatio gradatim ad 55°C pro 10~ 30min.
RT-LAMPA Protocol
| Components | Magnitudo | Finalis intentioni |
| Formula RNA | ad libitum | ≥10 copies |
| dNTP Mix (10mM) | 3.5 μL | 1.4 mM |
| FIP/BIP Primers (25×) | 1 μL | 1.6 μM |
| F3/B3 Primers (25×) | 1 μL | 0.2 μM |
| LoopF/LoopB Primers (25×) | 1 μL | 0.4 μM |
| RNAse Inhibitor (40U/μL) | 0.5 μL | XX U/Reactio |
| RTL Reverse Transcriptase 2.0 (15U/μL) | 0.5 μL | 7.5 U/Reactio |
| Bst V2 DNA Polymerase (8U/μL) | 1 μL | VIII U / reactionem |
| MgSO4 (100mM) | 1.5 μL | 6 mM (Summa 8 mM) |
| 10×HC RTL Buffer (vel 10×HC Bst V2 Buffer) | 2.5 μL | 1 (2mM Mg2+) |
| Nucleas-liberum aquae | Usque ad 25 μL | - |
Notae:
1) Miscere gurges et centrifuge breviter colligere.Assiduus temperatus incubatio 65°C pro 1 hora.
2) Duo buffers interoperabilia et eandem compositionem habent.
Notae
1. Productum hoc solidum faciet cum repositum ad -20 °C.Accipe eam ab -20°C et pone in glacie circiter X minutas.Postquam liquefactio adhiberi potest concutiendo et miscendo.
2. Productum cDNA sub -20°C vel -80°C condi potuit vel statim ad PCR reactionem adhibitum est.
3. RNase contagione prohibeat, quaeso ut area experimentalis munda et caestus munda et larvae in operatione induantur.
