Superstart qPCR Premix plus-UNG
Cat No: HCB5071E
Superstart qPCR Premix plus-UNG yog ib qho tshwj xeeb reagent tsim los rau Real Time PCR zoo thiab ntau cov tshuaj tiv thaiv siv kev soj ntsuam raws li kev tshawb nrhiav, tsim tshwj xeeb rau cov txheej txheem lyophilization.Nws muaj cov enzyme tshiab kub-pib Hotstart Taq ntxiv (DG), uas muaj nws cov Taq enzyme kev ua haujlwm kaw rau hauv chav tsev kub, ua haujlwm zoo inhibiting tsis tshwj xeeb amplification tshwm sim los ntawm primer non-specific annealing lossis primer dimer tsim nyob rau hauv qis kub, yog li txhim kho. qhov tshwj xeeb ntawm cov tshuaj tiv thaiv amplification.Cov reagent no siv qhov ua kom zoo dua qPCR tshwj xeeb tsis zoo thiab UNG / dUTP kev tiv thaiv kab mob kom ua tiav qhov pib kub sai, txhim kho kev ua tau zoo thiab rhiab heev ntawm qPCR cov tshuaj tiv thaiv.Nws tuaj yeem tau txais cov qauv nkhaus zoo hauv ntau thaj chaw kom muaj nuj nqis thiab ua kom muaj nuj nqis kom raug, tiv thaiv kev ua kom zoo tsis zoo los ntawm cov khoom PCR residual lossis aerosol paug.Cov reagent no tau sib xws nrog feem ntau cov khoom siv hluav taws xob fluorescent PCR los ntawm cov tuam txhab xws li Applied Biosystems, Eppendorf, Bio-Rad thiab Roche thiab lwm yam, thiab nthuav tawm kev ruaj ntseg zoo hauv daim ntawv lyophilized.
Reagent muaj pes tsawg leeg
1. 5 × HotstartPremix plus-UNG (Mg2+dawb) (DG)
2. 250 mM MgCl2
3. 4 × lyoprotectant (yeem)
Kev cia khoom
Lub sijhawm ntev cia ntawm -20 ℃;tuaj yeem khaws cia ntawm 4 ℃ txog li 3 lub hlis.Sib tov zoo ua ntej siv thiabtsis txhob rov khov thiab thawing.
Cycling Protocol
| Txheej txheem | Temp. | Sijhawm | Lub voj voog |
| Kev zom zaub mov | 50 ℃ | 2 min | 1 |
| Polymerase activation | 95 ℃ | 1 ~ 5 feeb | 1 |
| Denature | 95 ℃ | 10-20 s. : kuv | 40-50 |
| Annealing thiab Extension | 56 ~ 64 ℃ | 20-60 s. : kuv | 40-50 |
qPCR kua Reaction SyTxoj kev npaj
|
Kev sib xyaw |
25 µL ntim |
50 µL ntim |
Thaum kawg Concentration |
| 5 × HotstartPremix plus-UNG(Mg2+dawb) (DG) | 5 µL | 10 µL | 1 × |
| 250 mM MgCl2 | 0.45 l ua | 0,9l ua | 4,5m 0 |
| 4 × lyoprotectant1 | 6.25 l ua | 12.5 µL | 1 × |
| 25 × Primer-Probe Mix2 | 1 µL | 2 µL | 1 × |
| Template DNA3 | —— | —— | —— |
| ddH ua2O | Rau 25µL | Rau 50µL | —— |
1. Qhov kawg concentration ntawm 0.2μM rau primers feem ntau yields zoo;Thaum cov tshuaj tiv thaiv kev ua haujlwm tsis zoo, kho primer concentration nyob rau hauv thaj tsam ntawm 0.2-1μM raws li xav tau.Kev sojntsuam concentration yog feem ntau optimized nyob rau hauv thaj tsam ntawm 0.1-0.3μM los ntawm gradient thwmsim kom pom kev sib xyaw ua ke.
2. Cov ntawv luam ntawm cov hom phiaj muaj nyob hauv ntau hom qauv sib txawv;Yog tias tsim nyog, gradient dilution tuaj yeem ua tau los txiav txim siab qhov zoo tshaj plaws template ntxiv tus nqi.
3. Cov kab ke no tuaj yeem ua lyophilized;Thaum cov neeg siv khoom siv cov txheej txheem no yam tsis xav kom qhuav-qhuav, 4 × lyoprotectant tuaj yeem xaiv tau ntxiv; yog tias muaj cov khoom khov-qhuav yuav tsum tau, thaum lub sij hawm ua kua reagents theem cov khoom ua tau zoo, nws yuav tsum ntxiv 4 × lyoprotectant kom ntseeg tau tias muaj kev sib haum xeeb nrog cov khoom siv lyophilized. thiab cuam tshuam.
Thaum lub system sivd rau khov-kom qhuav, npaj cov qhov system as hauv qab no:
| Kev sib xyaw | 25µL Reaction System |
| 5 × HotstartPremix plus-UNG (Mg2+dawb) (DG) | 5 µL |
| 250 mM MgCl2 | 0.45 l ua |
| 4 × lyoprotectant | 6.25 l ua |
| 25 × Primer-Probe Mix | 1 µL |
| ddH ua2O | Rau 18 ~ 20µL |
* Yog tias xav tau lwm lub tshuab rau kev ziab kom qhuav, thov sab laj sib cais.
Lyophilization Txheej Txheemss
| Txheej txheem | Temp. | Sijhawm | Qhov xwm txheej | Siab |
| Ua ntej 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 ℃ | 70 feeb | Tuav | ||
| Secondary Drying | 25 ℃ | 60 feeb | Cua sov |
Qhov kawg Vacuum |
| 25 ℃ | 300 min | Tuav |
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 tseebtej yam kev mob thaum siv.
3. Cov txheej txheem lyophilization sib txawv yuav tsim nyog rau ntau qhov sib txawv ntawm lyophilizedcov khoom, yog li kev xeem txaus yuav tsum tau ua thaum siv rau kev tsim 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 dua ntawm qPCR.
2. Tsis muaj exogenous nuclease kev ua, tsis muaj exogenous endo/exonuclease paug.
Technical Information:
1. Superstart qPCR Premix plus-UNG siv cov enzyme tshiab kub-pib uas ua kom kub kub pib hauv 1 ~ 5 feeb;los ntawm kev tsim tshwj xeeb tsis yog nws tsim nyog rau multiplex fluorescent quantitative PCR cov tshuaj tiv thaiv.
2. Nws muaj qhov tshwj xeeb siab dua uas txhim kho qhov rhiab heev ntawm fluorescence quantitative PCR txwv kev kuaj pom, ua kom muaj qhov nkhaus nkhaus normalization, fluorescence tus nqi tau txais kev txhim kho pom tseeb ntawm cov qauv qis, haum raws li siab rhiab heev fluorescence quantitative PCR nrhiav reagents.
3. Rau primers nrog qis annealing kub los yog ntev tshaj 200bp fragments, 3-kauj ruam txoj kev yog pom zoo.
4. Kev siv qhov ua tau zoo ntawm dUTP thiab rhiab heev rau UNG enzyme txawv rau cov hom phiaj sib txawv, yog li yog siv UNG system ua rau txo qis hauv kev tshawb pom, cov tshuaj tiv thaiv yuav tsum tau kho thiab ua kom zoo dua.Yog xav tau kev txhawb nqa thov hu rau peb lub tuam txhab.
5. Siv cov chaw tshwj xeeb thiab cov kav dej ua ntej thiab tom qab kev nthuav dav, hnav cov hnab looj tes thaum ua haujlwm thiab hloov lawv ntau zaus;tsis txhob qhib lub raj tshuaj tiv thaiv tom qab ua tiav PCR kom txo tau cov kab mob sib kis los ntawm cov khoom PCR.
