5 × Neoscript Fast RT-qPCR Premix plus-UNG
Cat No: HCB5142A
Neoscript Fast RT Premix-UNG (Probe qRT-PCR) yog ib qho ruaj khov ib-tube sojntsuam-raws li sib xyaw haum rau ib-kauj ruam thim rov qab thiab ntau PCR (qRT-PCR).Nws txhawb kev sib xyaw ua ntej ntawm primers thiab probes thiab nyob ruaj khov tom qab lub sijhawm ntev cia ntawm qhov kub thiab txias.Cov qauv uas yuav tsum tau sim tuaj yeem muab ntxiv ncaj qha thaum siv, tsis tas siv lub raj qhib / pipeetting.Cov khoom no muab cov khoom xyaw, xws li kub-pib DNA polymerase, M-MLV, tshav kub-labile uracil DNA glycosylase (TS-UNG), RNase Inhibitor, MgCl2, dNTPs (nrog dUTP hloov dTTP), thiab stabilizers.Nrog rau kev hloov kho caj ces nrawm nrawm rov qab transcriptase thiab DNA polymerase, nws muaj peev xwm ua kom tiav PCR amplification hauv 20-40 feeb.Cov reagent no siv tshwj xeeb tsis pub rau qPCR nrog cov enzymes sib xyaw ntawm cov tshuaj tiv thaiv kev ua kom tsis muaj zog thiab UNG enzyme.Yog li ntawd, nws tuaj yeem tau txais txiaj ntsig zoo ntawm lub hom phiaj cov noob thiab tiv thaiv kev ua kom tsis muaj zog los ntawm PCR residual thiab aerosol pollution.Cov reagent no tau sib xws nrog feem ntau fluorescence quantitative PCR cov cuab yeej los ntawm cov tuam txhab xws li Applied Biosystems, Eppendorf, Bio-Rad thiab Roche.
Cheebtsam
1.25 × Neoscript Fast RTase/UNG Mix
2.5 × Neoscript Fast RT Premix Buffer (dUTP)
Kev cia khoom
Tag nrho cov khoom yuav tsum tau khaws cia ntawm -20 ℃ rau kev cia ntev thiab 4 ℃ txog li 3 lub hlis.Thov sib tov kom huv si tom qab thawing thiab centrifuge ua ntej siv.Tsis txhob nquag khov-thaw.
qRT-PCR Reaction System Npaj
| Cheebtsam | 25μLQhov system | 50μLQhov system | Thaum kawg Concentration |
| 5 × Neoscript Fast RT Premix Buffer (dUTP) | 5l miv | 10 mL | 1 × |
| 25 × Neoscript Fast RTase/UNG Mix | 1 m l | 2 m l | 1 × |
| 25 × Primer-Probe Mixa | 1 m l | 2 m l | 1 × |
| Template RNAb | - | - | - |
| ddH ua2O | Txog li 25μL | Txog li 50μL | - |
1) a.Qhov kawg concentration ntawm primer feem ntau yog 0.2μM.Rau cov txiaj ntsig zoo dua, primer concentration tuaj yeem ua kom zoo nyob rau hauv thaj tsam ntawm 0.2-1μM.Feem ntau, qhov kev sojntsuam concentration tuaj yeem ua kom zoo nyob rau hauv thaj tsam ntawm 0.1-0.3μM.
2) b.Thaum siv cov txheej txheem PCR ceev, ua kom cov concentration ntawm primers thiab sojntsuam yuav ua rau kom zoo dua amplification, thiab lawv cov piv yuav tsum optimized raws li.
3) Cov qauv sib txawv muaj ntau hom thiab cov ntsiab lus ntawm inhibitor thiab luam tus naj npawb ntawm cov hom phiaj.Cov qauv ntim yuav tsum tau txiav txim siab los ntawm qhov tseeb.Ua kom dilution ntawm tus qauv nrog cov dej tsis muaj nuclease lossis TE Buffer, yog tias tsim nyog.
Teb Chaws Ckev xav
| Cov txheej txheem PCR tsis tu ncua | Ceev ceev PCR txheej txheem | ||||||
| Txheej txheem | Temp. | Sijhawm | Lub voj voog | Txheej txheem | Temp. | Sijhawm | Lub voj voog |
| Rov qab Transcription | 50 ℃ | 10-20 feeb | 1 | Rov qab Transcription | 50 ℃ | 5 feeb | 1 |
| Polymerase Ua kom | 95 ℃ | 1-5 feeb | 1 | Polymerase Ua kom | 95 ℃ | 30s ib | 1 |
| Denaturation | 95 ℃ | 10-20s | 40-50 | Denaturation | 95 ℃ | 1-3 s | 40-50 |
| Annealing thiab Txuas ntxiv | 56-64 ℃ | 20-60s | Annealing thiab Txuas ntxiv | 56-64 ℃ | 3-20s | ||
Kev Tswj Xyuas Zoo
1.Kev kuaj pom muaj nuj nqi: rhiab heev, tshwj xeeb thiab rov ua dua ntawm qPCR.
2.Tsis muaj exogenous nuclease kev ua: tsis muaj exogenous endonuclease thiab exonuclease muaj kuab paug.
Sau ntawv
1.Amplification tus nqi ntawm DNA polymerase ceev yog tsis tsawg tshaj li 1kb/10s.Cov cuab yeej PCR sib txawv muaj qhov sib txawv cua sov thiab txias ceev, hom kev tswj kub thiab thermal conductivity, thiab yog li kev ua kom zoo ntawm koj cov primer / sojntsuam concentration thiab khiav txoj kev ua ke nrog koj cov cuab yeej PCR tshwj xeeb yog qhov tseem ceeb.
2.Cov khoom no ua haujlwm dav dav, thiab nws tsim nyog rau kev kuaj mob siab molecular.Peb-kauj ruam PCR txoj kev pom zoo rau cov primers uas tsis tshua muaj annealing kub los yog rau amplification ntawm ntev fragments tshaj 200 bp.
3.Txij li cov amplicons sib txawv muaj qhov sib txawv ntawm kev siv dUTP thiab sib txawv rhiab heev rau UNG, cov reagents yuav tsum tau ua kom zoo dua yog tias qhov kev tshawb pom rhiab heev txo qis thaum siv UNG system.Thov hu rau peb rau kev txhawb nqa yog tias xav tau.
4.Txhawm rau zam kom tsis txhob muaj kev nthuav dav ntawm cov khoom nqa PCR, thaj chaw sim thiab cov kav dej yog xav tau rau kev nthuav dav.Ua haujlwm nrog cov hnab looj tes thiab hloov ntau zaus thiab tsis txhob qhib lub PCR raj tom qab kev nthuav dav.
