2 × Sensi Direct Premix-UNG (Kev sojntsuam qPCR)
Cat No: HCB5151A
SensiDirect Premix-UNG (Probe qPCR) yog tsim los ua PCR ncaj qha los ntawm cov qauv yam tsis muaj DNA rho tawm lossis qauv npaj.Cov reagent no muaj kub pib DNA polymerase, uracil DNA glycosylase (UNG), RNase Inhibitor, MgCl2, dNTPs (nrog dUTP es tsis txhob dTTP), thiab stabilizers, rau quantitative PCR (qPCR).Qhov no reagent preforms siab inhibitor-kev ua siab ntev, thiab yog li nws tuaj yeem siv ncaj qha rau kev kuaj pom xws li caj pas, qaub ncaug, tiv thaiv cov ntshav tag nrho, ntshav ntshav, thiab ntshav tsis muaj DNA rho tawm.Cov reagent siv cov khoom tsis pub rau qPCR nrog cov enzymes sib xyaw ntawm anti-inhibitory DNA polymerase thiab UNG enzyme.Yog li ntawd, nws tuaj yeem tau txais kev nthuav dav zoo ntawm cov hom phiaj hauv cov qauv uas muaj cov tshuaj inhibitors thiab inhibit qhov tsis zoo ntawm qhov ua kom pom tseeb los ntawm PCR residual thiab aerosol pollution.Cov reagent no tau sib xws nrog cov khoom siv fluorescent ntau PCR, xws li Applied Biosystems, Eppendorf, Bio-Rad, Roche thiab lwm yam.
Cheebtsam
1. 50 × SensiDirect Enzyme/UNG Mix
2. 2 × SensiDirect Premix Buffer (dUTP)
Cia tej yam kev mob
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.
Cycling Protocol
| Kauj ruam | Kub | Sijhawm | Lub voj voog |
| Kev zom zaub mov | 50 ℃ | 2 min | 1 |
| Polymerase activation | 95 ℃ | 1-5 feeb | 1 |
| Denature | 95 ℃ | 10-20s | 40-50 |
| Annealing / Extension | 56-64 ℃ | 20-60s |
Cov lus qhia Pipetting
| Reagent | Volume per tshuaj tiv thaiv | Volume rau cov tshuaj tiv thaiv | Thaum kawg Concentration |
| 2 × SensiDirect Premix Buffer (dUTP) | 12.5 µL | 25 µL | 1 × |
| 50 × SensiDirect Enzyme/UNG Mix | 0.5 l ;ua | 1 µL | 1 × |
| 25 × Primer-Probe Mix1, 2 | 1 µL | 2 µL | 1 × |
| Qauv3, 4 | - | - | - |
| ddH ua2O | - | - | - |
| Tag nrho ntim | 25l miv | 50l ua | - |
1. 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.
2. Feem ntau, kev sojntsuam concentration tuaj yeem ua kom zoo nyob rau hauv thaj tsam ntawm 0.1-0.3μM.Qhov zoo tshaj plaws concentration ntawm kev sojntsuam yog muaj feem xyuam rau lub sij hawm ntawm PCR amplification ntsuas, hom kev sojntsuam, thiab hom tshuaj fluorescent labeling.Thov xa mus rau phau ntawv ntsuas ntsuas lossis cov kev cai tshwj xeeb ntawm txhua qhov kev sojntsuam fluorescent.
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 los ntawm kev ntxiv cov dej tsis muaj nuclease lossis TE Buffer, yog tias tsim nyog.
4. Pom zoo ntim ntawm cov qauv sib txawv:
| Qauv | Volume rau ib 50 μL tshuaj tiv thaiv | Qhov siab tshaj plaws piv |
| Anticoagulated tag nrho cov ntshav | 2.5 l ua | 5% |
| Plasma | 15 mL | 30% |
| Serum | 10 mL | 20% |
| caj pas swab | 10 mL | 20% |
| Kua qaub | 10 mL | 20% |
Kev Tswj Xyuas Zoo
1. Kev tshawb nrhiav 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.
Cov Lus Cim ntawm Cov Khoom
1. Cov khoom no siv ib hom tshiab ntawm kub-pib DNA polymerase, uas tuaj yeem qhib tau hauv 1-5 feeb.Vim nws cov tshuaj tiv thaiv tsis tau raug kho kom zoo, nws yog qhov tsim nyog rau ob lossis ntau lub fluorescence quantitative PCR siv txoj kev sojntsuam.
2. Yog hais tias tus nqi Rn ntawm PCR amplification tsawg dhau los yog qhov amplification yog pom tseeb inhibited, txo tus nqi piv txwv, nce cov tshuaj tiv thaiv los yog yav dhau los dilution ntawm cov qauv yuav txhim kho cov txiaj ntsig.
3. Kev sau cov ntshav, qaub ncaug, zis, caj pas, thiab lwm yam yuav tsum ua raws li cov txheej txheem kho mob, thiab cov qauv tshiab tuaj yeem siv los tiv thaiv nucleic acid degradation.
4. Vim tias cov amplicons sib txawv muaj qhov sib txawv ntawm kev siv rau dUTP thiab 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.
5. Txhawm rau kom tsis txhob muaj kev nthuav dav ntawm cov khoom nqa PCR ntawm ib-kauj ruam cov tshuaj tiv thaiv, thaj chaw sim thiab cov pipette yog xav tau rau kev nthuav dav.Ua haujlwm nrog cov hnab looj tes thiab hloov ntau zaus thiab tsis txhob qhib lub raj tshuaj tiv thaiv tom qab PCR amplification.
