I-Multiplex One Step RT-qPCR Premix-UNG
Inombolo yekati: HCB5145A
I-Multiplex One Step RT-qPCR Probe Kit (UDG Plus) iyikhithi ye-PCR yobuningi be-multiplex esekelwe ku-RNA njengesifanekiso.Enqubweni yokuhlolwa, ukuloba okuhlanekezelwe kanye ne-PCR yobuningi kwenziwa ngeshubhu efanayo, eyenza umsebenzi wokuhlola waba lula futhi yehlisa ubungozi bokungcola.Kule khithi, i-cDNA yokuqala yomucu yahlanganiswa kahle yi-Reverse Transcriptase engashi futhi yakhuliswa ngobuningi yi-HotStart Tag DNA Polymerase.Ikhithi ikakhulukazi iqukethe i-MP buffer elungiselelwe kahle, inhlanganisela yama-enzyme, njll. Isixazululo sebhafa sesivele sine-Mg.2+kanye ne-dNTP.Ngaphezu kwalokho, izinto ezingavimbela ngempumelelo ukukhulisa i-PCR okungaqondile futhi zithuthukise ukusebenza kahle kokukhulisa ukusabela okuningi kwe-qPCR ziyengezwa, ezingaqinisekisa ukusebenza kahle kokukhulisa futhi zenze ukusabela kokukhulisa okuningi.Uhlelo lwe-dUTP/UDG lwengezwe ukuze kuvinjelwe ngempumelelo ubungozi bokungcoliswa kwe-aerosol.
Izingxenye
1. Ibhafa
2. I-Enzyme Mix
Ukucaciswa
| Isiqalo Esishisayo | Isiqalo esishisayo esakhelwe ngaphakathi |
| Indlela yokuthola | Ukutholwa kwe-Primer-probe |
| Indlela ye-PCR | Isinyathelo esisodwa RT-qPCR |
| I-Polymerase | Taq DNA polymerase |
| Uhlobo lwesampula | I-DNA |
Izimo Zokugcina
Umkhiqizo uthunyelwa ngeqhwa elomile futhi ungagcinwa ku -25 ~ -15 ℃ unyaka ongu-1.Kufanele igweme njaloqhwa-ncibilika.Kunconywa ukulondoloza ngokwehlukana.
Iziyalezo
1.Ukusabela Uhlelo
| Izingxenye | Ivolumu (μL) | Ukugxila Kokugcina |
| 2 × I-MP Buffer | 12.5 | 1× |
| I-Enzyme Mix | 1 | - |
| Ingxube ye-Primer/Probe (2.5 μM) | 3 | 0.3μM |
| I-RNA yesifanekiso | 1-10 | - |
| RNase Mahhala H2O | kuye 25 | - |
Amanothi:
Qinisekisa ukuthi uxuba kahle ngaphambi kokusebenzisa, gwema amabhamuza amaningi adalwa ukudlidliza okunodlame.
a.I-Primer concentration: I-Primer mix ehlanganisa i-multiplex primer, kuye ngesimo sokugxiliswa kwe-primer elungile mhlawumbe phakathi kuka-0.l no-1.0μM.
b.Ukugxiliswa kwe-probe: Ingxube ye-probe ehlanganisa iqembu le-multiplex probe elebula umehluko we-fluorescent, kuye ngesimo sokugxiliswa kwe-probe okungenzeka phakathi kuka-0.05 no-0.5μM.
c.Ukuhlanjululwa kwesifanekiso: I-qPCR izwela kakhulu futhi kuyanconywa ukuthi kuhlanjululwe isifanekiso.Inani le-Ct lokulawula lifanele phakathi kuka-20 no-35.
d.Ukulungiswa kwesistimu: Sicela ulungiselele etafuleni lokusebenza elihlanzekile kakhulu, kanye ne-pipettor kanye neshubhu yokusabela ngaphandle kwezinsalela ze-nuclease;kunconywa ukusebenzisa ikhanda lesibhamu elinesici sokuhlunga.Gwema ukungcola okuphambanayo kanye nokungcoliswa kwe-aerosol.
2. Ukuhamba Ngebhayisikili OkuthuthukisiweIphrothokholi
| Umjikelezo isinyathelo | Temp. | Isikhathi | Imijikelezo |
| Hlehlisa ukuloba | 50℃a | 20 imiz | 1 |
| I-denaturation yokuqala | 95℃ | 5 imiz | 1 |
| Ukusabela kokukhulisa | 95℃ | 15 isekhondi |
40-45 |
| 60℃ b | 30 isekhondi c |
Amanothi:
a.Ukuhlehlisa okulotshiweyo: Izinga lokushisa lingakhetha u-42°C noma u-50°C.
b.Ukusabela kwe-Amplification: Izinga lokushisa lilungiswa ngokuvumelana nenani le-Tm lama-primer aklanyelwe.
c.Ukutholwa kwesiginali ye-Fluorescence:Sicela usethe inqubo yokuhlola ngokuya ngezidingo zemanuwali yezinsimbi.
Amanothi
Sicela ugqoke i-PPE edingekayo, njengejazi laselabhu namagilavu, ukuze uqinisekise impilo nokuphepha kwakho.
