Fast Ampli RT-qPCR Premix plus-UNG
Cat No: HCB5144E
Fast Ampli RT-qPCR Premix plus-UNG yog tsim tshwj xeeb rau cov txheej txheem lyophilization, thov rau fluorescence quantification (TaqMan sojntsuam) RNA amplification nrhiav uas muaj neocript thim rov qab transcriptase thiab ceev amplification DNA Polymerase tau los ntawm genetic hloov thiab tshuaj ntsuam xyuas uas tuaj yeem ua tiav hauv PCR amplification. -40 feeb.Qhov no reagent preforms siab inhibitor- kam rau ua, uas muaj ntau dua reverse transcription thiab PCR amplification efficiency, haum rau high-rhiab heev amplification ntawm low-concentration RNA qauv.Ib qho qauv nkhaus zoo tuaj yeem tau txais nyob rau hauv ntau qhov ntau thiab tsawg, thiab kev ua kom muaj nuj nqis tuaj yeem ua tiav.Qhov no reagent siv ib tug sib xyaw enzyme ntawm anti-inhibition amplification enzyme thiab UNG enzyme, raws li zoo raws li ib tug optimized buffer system uas muaj dUTP.Nws tuaj yeem ua tsis tau tsuas yog ua tau zoo amplification ntawm lub hom phiaj gene nyob rau hauv cov qauv uas muaj inhibitors, tab sis kuj zoo tiv thaiv tsis zoo amplification tshwm sim los ntawm PCR residual thiab aerosol pa phem.Cov reagent no tau sib xws nrog cov khoom siv fluorescent ntau PCR, xws li Applied Biosystems, Eppendorf, Bio-Rad, Roche thiab lwm yam.Cov reagent no tuaj yeem siv rau lyophilization kom tau txais daim ntawv lyophilized zoo thiab khoom ruaj khov.
Reagent muaj pes tsawg leeg
1. 12.5 × FastAmpli Part Taq/UNG Mix (nrog dNTPs) (DG)
2. 50 × FastAmpli Part RTase (DG)
3. 5 × FastAmpliRT Buffer (DG)
4. 4 × lyoprotectant (yeem) (DG)
Kev cia khoom
Nws tuaj yeem khaws cia ntawm -20 ℃ rau lub sijhawm ntev, ntawm 4 ℃ txog li 3 lub hlis.Sib tov zoo ua ntej siv thiab zamnquag khov-thaw.
Cycling Protocol
|
Kauj ruam |
Kub | Tsis tu ncua PCR Txheej txheem | Fast PCRTxheej txheem |
Lub voj voog |
| Duration | Duration | |||
| Rov qabKev sau ntawv | 50 ℃ | 15 feeb | 5 feeb | tuav |
| PolymeraseUa kom | 95 ℃ | 1-5 feeb | 1-2 feeb | tuav |
| Denature | 95 ℃ | 10-20 s., kuv | 1-3 sec | 40-50 |
| Annealing / Extension | 56-64 ℃ | 20-60 s., kuv | 3-20 sec |
RT-qPCR kua Reaction System
| Kev sib xyaw | 25 µL Ntim | 50 µL Ntim | Nco ntsoov |
| 5 × FastAmpli RT Buffer | 5 µL | 10 µL | 1 × |
| 12.5 × FastAmpli Part Taq/UNG Mix (nrog dNTPs) | 2 µL | 4 µL | 1 × |
| 50 × FastAmpli Part RTase | 0.5 l ;ua | 1 µL | 1 × |
| 4 × lyoprotectant | 6.25 l ua | 12.5 µL | 1 × |
| 25 × Primer-Probe Mix | 1 µL | 2 µL | 1 × |
| Template RNA | — | — | — |
| ddH ua2O | Rau 25µL | Rau 50µL | 1 × |
1. Cov kab ke no yog qhov system lyophilization;Thaum cov neeg siv khoom siv qhov system no yam tsis tas yuav tsum muaj lyophilization, 4 × lyoprotectant tuaj yeem xaiv tau ntxiv;Yog tias muaj cov khoom lag luam lyophilized yuav tsum tau, thaum lub sij hawm ua kua reagents theem khoom validation, nws yuav tsum ntxiv 4 × lyoprotectant los xyuas kom meej sib xws nrog cov lyophilized system Cheebtsam thiab cov teebmeem.
2. Thaum siv cov txheej txheem PCR tsis tu ncua, qhov kawg concentration ntawm primer feem ntau yog 0.2μM.Rau cov txiaj ntsig zoo dua, cov primer concentration tuaj yeem ua kom zoo nyob rau hauv thaj tsam ntawm 0.2- 1.0μM.Kev sojntsuam concentration tuaj yeem ua kom zoo nyob rau hauv thaj tsam ntawm 0.1-0.3μM.Concentration gradient thwmsim tuaj yeem ua tau los nrhiav qhov zoo tshaj plaws ua ke ntawm primers thiab probes.
3. Thaum siv cov txheej txheem ceev PCR amplification, ib qho txiaj ntsig zoo tuaj yeem ua tiav los ntawm kev nce primer / sojntsuam concentration, nrog rau kev kho qhov feem pua ntawm primer thiab sojntsuam.
4. Cov qauv sib txawv muaj cov qauv sib txawv ntawm cov hom phiaj.Ib qho gradient dilution tuaj yeem ua tiav los xaiv qhov tsim nyog zoo template ntxiv tus nqi, yog tias tsim nyog.
Lyophilization system npaj
| Kev sib xyaw | 25µL Cov tshuaj tiv thaiv Qhov system |
| 5 × FastAmpli RT Buffer | 5 µL |
| 12.5 × FastAmpli Part Taq/UNG Mix (nrog dNTPs) (DG) | 2 µL |
| 50 × FastAmpli Part RTase (DG) | 0.5 l ;ua |
| 4 × Lyoprotectant | 6.25 l ua |
| 25 × Primer-Probe Mix | 1 µL |
| ddH ua2O | Rau 18 ~ 20µL |
* Yog xav tau lwm lub tshuab rau lyophilization, thov sab laj sib cais.
Lyophilization Proces
| Txheej txheem | Temp. | Sijhawm | Qhov xwm txheej | Siab |
| khov | 4 ℃ | 30 feeb | Tuav | 1 awm |
| -50 ℃ | 60 feeb | Txias | ||
| -50 ℃ | 180 feeb | Tuav | ||
| Kev ziab qhuav | -30 ℃ | 60 feeb | Cua sov | Qhov kawg Vacuum |
| -30 ℃ | 720 nqi | Tuav | ||
| Secondary Drying | 25 ℃ | 60 feeb | Cua sov | Qhov kawg Vacuum |
| 25 ℃ | 300 min | Tuav |
1. Cov txheej txheem lyophilization no yog txheej txheem nyob rau hauv-situ freeze-drying rau 25µL cov tshuaj tiv thaiv kab mob;yog tias khov-drying hlaws los yog lwm yam hauv-situ freeze-drying txheej txheem yuav tsum tau, thov nug cais.
2. Cov txheej txheem lyophilization saum toj no yog rau kev siv xwb.Cov khoom sib txawv thiab cov tshuab ziab khaub ncaws sib txawv muaj qhov sib txawv, yog li kev hloov kho tuaj yeem ua raws li qhov tseeb thaum siv.
3. Cov txheej txheem lyophilization sib txawv tuaj yeem tsim nyog rau ntau qhov sib txawv ntawm cov khoom sib txawv lyophilized, yog li kev ntsuas txaus yuav tsum tau ua thaum siv rau cov khoom loj.
Cov lus qhia rau kev siv lyophilized hmoov
1. Luv luv centrifuge cov lyophilized hmoov;
2. Ntxiv nucleic acid template rau lyophilized hmoov thiab ntxiv dej mus txog 25µL;
3. Sib tov zoo los ntawm centrifugation thiab khiav ntawm lub tshuab.
Kev Tswj Xyuas Zoo
1. Kev sim ua haujlwm: rhiab heev, tshwj xeeb, rov tsim tawm ntawm RT-qPCR.
2. Tsis muaj exogenous nuclease kev ua, tsis muaj exogenous endo/exonuclease paug.
Technical Information:
1. Tus nqi amplification ntawm ceev DNA polymerase 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, yog li kev ua kom zoo ntawm koj cov primer / sojntsuam concentration thiab khiav txoj hauv kev ua ke nrog koj cov cuab yeej PCR tshwj xeeb yog qhov tseem ceeb.
2. Lub sij hawm thim rov qab yog optimized raws li qhov ntev ntawm cov fragment kom amplified.Rau cov ntu ntev dua, lub sijhawm rov qab hloov pauv tuaj yeem txuas ntxiv kom tsim nyog.
3. Lub annealing extension kub yuav tsum tau optimized raws li tus nqi Tm ntawm primer sojntsuam thiab qhov tseeb tej yam kev mob ntawm cov tshuaj tiv thaiv.
4. Rau primers nrog qis annealing kub los yog ntev tshaj 200 bp fragments, 3-kauj ruam txoj kev yog pom zoo.
5. Thov siv cov chaw tshwj xeeb thiab cov kav dej ua ntej thiab tom qab kev nthuav dav, hnav cov hnab looj tes thiab hloov ntau zaus;tsis txhob qhib lub raj tshuaj tiv thaiv tom qab ua tiav cov tshuaj tiv thaiv PCR kom txo tau cov kab mob sib kis los ntawm cov khoom PCR.
6. Kev siv tau zoo ntawm dUTP thiab rhiab heev rau UNG enzyme txawv rau cov hom phiaj sib txawv, yog li yog tias siv UNG system ua rau txo qis hauv kev tshawb pom, cov tshuaj tiv thaiv yuav tsum tau kho thiab kho kom zoo.Yog xav tau kev txhawb nqa thov hu rau peb.
