RTL Reverse Transcriptase Cov Lus Qhia
RTL thim rov qab transcriptase yog RNA template-dependent DNA polymerase uas tsis muaj 3'→ 5' exonuclease kev ua thiab muaj RNase H ua haujlwm.Cov enzyme no tuaj yeem siv RNA los ua tus qauv los tsim cov kab sib txuas ntxiv ntawm DNA, uas tuaj yeem siv rau thawj-strand cDNA synthesis, tshwj xeeb tshaj yog rau RT-LAMP (loop-mediated isothermal amplification).Piv nrog RTL thim rov qab transcriptase 1.0, qhov rhiab heev yog txhim kho, thermal stability muaj zog dua, thiab cov tshuaj tiv thaiv ntawm 65 ° C yog ruaj khov dua.RTL thim rov qab transcriptase (glycerol dawb) tuaj yeem siv los npaj cov tshuaj lyophilized, lyophilized RT-LAMP reagents thiab lwm yam.
Unit txhais
Ib chav tsev suav nrog 1 nmol ntawm dTTP rau hauv cov khoom siv acid-precipitable hauv 20 feeb ntawm 50 ° C siv poly(A) •oligo(dT)25 ua template-primer.
Cheebtsam
| Cheebtsam | TSI HC5008A-01 | TSI HC5008A-02 | TSI HC5008A-03 |
| RTL Reverse Transcriptase (Glycerol-dawb) (15U / μL) | 0.1ml ib | 1 ml ib | 10 ml ib |
| 10 × HC RTL Tsis | 1.5 ml yog ' | 4 × 1.5 mL | 5 × 10 ml |
| MgSO4 (100mM) | 1.5 ml yog ' | 2 × 1.5 mL | 3 × 10 ml |
Kev cia khoom
Kev thauj mus los hauv qab 0 ° C thiab khaws cia ntawm -25 ° C ~ -15 ° C.
Kev Tswj Xyuas Zoo
- Kev ua haujlwm seem ntawmEndonuclease:Ib qho 50 μL cov tshuaj tiv thaiv uas muaj 1 μg ntawm λDNA thiab 15 units ntawm RTL2.0 incubated rau 16 teev ntawm 37 ℃ qhia tib tus qauv raws li kev tswj tsis zoo los ntawm gel electrophoresis.
- Kev ua haujlwm seem ntawmExonuclease:Ib 50 μL cov tshuaj tiv thaiv uas muaj 1 μg ntawm Hind Ⅲ digested λDNA thiab 15 units ntawm RTL2.0 incubated rau 16 teev ntawm 37 ℃ qhia tib tus qauv raws li kev tswj tsis zoo los ntawm gel electrophoresis.
- Kev ua haujlwm seem ntawmNickase:Ib qho 50 μL cov tshuaj tiv thaiv uas muaj 1 μg ntawm supercoiled pBR322 thiab 15 units ntawm RTL2.0 incubated rau 4 teev ntawm 37 ° C qhia tib yam li kev tswj tsis zoo los ntawm gel electrophoresis.
- Kev ua haujlwm seem ntawmRNase:Ib qho 10 μL cov tshuaj tiv thaiv uas muaj 0.48 μg ntawm MS2 RNA thiab 15 units ntawm RTL2.0 incubated rau 4 teev ntawm 37 ° C qhia tib yam li kev tswj tsis zoo los ntawm gel electrophoresis.
- E. coli gDNA:Ntsuas nrogE.coliCov khoom siv kuaj HCD tshwj xeeb, 15 units ntawm RTL2.0 muaj tsawg dua 1E. coligenome.
Kev teeb tsa kev daws teeb meem
cDNA Synthesis raws tu qauv
| Cheebtsam | Ntim |
| Template RNA ib | xaiv tau |
| Oligo (dT) 18 ~ 25 (50uM) los yog Random Primer mix (60uM) | 2l miv |
| dNTP Mix (10mM txhua) | 1 mL ib |
| RNase Inhibitor (40U/uL) | 0,5l ua |
| RTL Cov Ntawv Tshaj Lij Tshaj Tawm 2.0 (15U / uL) | 0,5l ua |
| 10 × HC RTL Tsis | 2l miv |
| Dej tsis muaj Nuclease | Txog li 20 μL |
Nco tseg:
1) Kev pom zoo noj ntawm Tag Nrho RNA yog 1ng ~ 1μg
2) Cov tshuaj pom zoo ntawm mRNA yog 50ng ~ 100ng
Thermo-cycling Cov xwm txheej rau kev niaj hnub tshuaj tiv thaiv:
| Qhov kub thiab txias (°C) | Sijhawm |
| 25 ° Ca | 5 feeb |
| 55 ° C | 10 feebb |
| 80 ° C | 10 feeb |
Nco tseg:
1) Yog siv Random Primer Mix, ib kauj ruam incubation ntawm 25 ° C.
2) Yog hais tias lub hom phiaj primer mix yog siv, ib kauj ruam incubation ntawm 55 ° C rau 10 ~ 30mins.
RT-LAMP raws tu qauv
| Cheebtsam | Ntim | Thaum kawg Concentration |
| Template RNA | xaiv tau | ≥10 daim ntawv |
| dNTP sib tov (10mM) | 3,5l ua | 1,4m wb |
| FIP / BIP Primers (25 ×) | 1 mL ib | 1.6m ua |
| F3 / B3 Primers (25 ×) | 1 mL ib | 0,2m ua |
| LoopF/LoopB Primers (25 ×) | 1 mL ib | 0,4m wb |
| RNase Inhibitor (40U / μL) | 0,5l ua | 20 U/Reaction |
| RTL Cov Ntawv Tshaj Tawm 2.0 (15U / μL) | 0,5l ua | 7.5 U/Reaction |
| Bst V2 DNA Polymerase (8U/μL) | 1 mL ib | 8 U/Reaction |
| MgSO4 (100mM) | 1.5 l ua | 6 mM (tag nrho 8 mM) |
| 10 × HC RTL Buffer (los yog 10 × HC Bst V2 Buffer) | 2.5 l ua | 1 × (2mM Mg2+) |
| Dej tsis muaj Nuclease | Txog li 25 μL | - |
Nco tseg:
1) Sib tov los ntawm vortexing thiab centrifuge luv luv los sau.Qhov kub thiab txias incubation ntawm 65 ° C rau 1 teev.
2) Ob lub buffers sib cuam tshuam thiab muaj qhov sib xws.
Sau ntawv
1.Cov khoom no yuav tsim cov khoom dawb thaum khaws cia ntawm -20 ° C.Tshem tawm ntawm -20 ° C thiab muab tso rau hauv dej khov li 10 feeb.Tom qab melting, nws tuaj yeem siv los ntawm kev co thiab sib tov.
2.Cov khoom cDNA tuaj yeem khaws cia ntawm -20 ° C lossis -80 ° C lossis siv tam sim rau PCR cov tshuaj tiv thaiv.
3. Txhawm rau tiv thaiv kev kis kab mob RNase, thov khaws thaj chaw sim kom huv si, thiab hnav hnab looj tes huv si thiab lub qhov ncauj qhov ntswg thaum ua haujlwm.
