Multiplex Inyathelo elinye RT-qPCR Premix-UNG
Inombolo yekati: HCB5145A
I-Multiplex One Step RT-qPCR Probe Kit (UDG Plus) yikiti ye-PCR ephindaphindayo esekelwe kwi-RNA njenge template.Kwinkqubo yovavanyo, ukukhutshelwa umva kunye ne-PCR yobuninzi benziwa kwityhubhu efanayo, eyenza lula umsebenzi wokulinga kunye nokunciphisa umngcipheko wokungcola.Kule khithi, i-cDNA yokuqala yomtya yadityaniswa ngokufanelekileyo yi-Reverse Transcriptase ekwazi ukumelana nobushushu kwaye yandiswa ngokobungakanani yi-HotStart Tag DNA Polymerase.Ikhithi iqulathe ikakhulu isithinteli seMP, umxube weeenzymes, njalo njalo.2+kunye ne-dNTP.Ukongeza, izinto ezinokuthi zithintele ngokufanelekileyo ulwandiso lwe-PCR olungacacisiweyo kunye nokuphucula ukusebenza kakuhle kolwandiso lweempendulo ezininzi ze-qPCR zongezwa, ezinokuqinisekisa ukusebenza kakuhle kokukhulisa kwaye ziqhubele phambili ukusabela kokukhulisa okuninzi.Inkqubo ye-dUTP/UDG yongezwa ukuthintela ngokufanelekileyo umngcipheko wongcoliseko lwe-aerosol.
Amacandelo
1. Isithinteli
2. Umxube we-Enzyme
Inkcazo
| Isiqalo esishushu | Isiqalo esishushu esakhelwe ngaphakathi |
| Indlela yokubona | Ukufunyanwa kwe-Primer-probe |
| Indlela yePCR | Inyathelo elinye RT-qPCR |
| Ipolymerase | Taq DNA polymerase |
| Uhlobo lwesampulu | DNA |
Iimeko zokuGcina
Imveliso ithunyelwa ngeqhwa elomileyo kwaye ingagcinwa kwi -25 ~ -15 ℃ ngonyaka we-1.Kufuneka kuphephe rhoqongumkhenkce-nyibilika.Kucetyiswa ukuba ugcine ngokwahlukileyo.
Imiyalelo
1.Ukusabela Inkqubo
| Amacandelo | Umthamo (μL) | Ukugxininiswa kokugqibela |
| I-2 × i-MP Buffer | 12.5 | 1× |
| Umxube we-Enzyme | 1 | - |
| I-Primer/Probe mix (2.5 μM) | 3 | 0.3μM |
| Ithempleyithi ye-RNA | 1-10 | - |
| RNase Mahala H2O | ukuya ku25 | - |
Amanqaku:
Qinisekisa ukuba uxuba kakuhle phambi kokusetyenziswa, thintela amaqamza agqithisileyo abangelwa kukungcangcazela okunobundlobongela.
a.I-Primer concentration: I-Primer mix equka i-multiplex primer, ngokuxhomekeke kwimeko yogxininiso lweprimer olugqwesileyo mhlawumbi phakathi kwe-0.l kunye ne-1.0μM.
b.Uxinaniso lweProbe: Umxube weProbe uquka ukuphinda-phinda ukuleyibhela kweqela leprobe umahluko, kuxhomekeke kwimeko yoxinaniso lweprobe mhlawumbi phakathi kwe-0.05 kunye ne-0.5μM.
c.I-Template dilution: I-qPCR inovakalelo oluphezulu kwaye kuyacetyiswa ukuba udibanise itemplate.Ixabiso le-Ct lolawulo lifanelekile phakathi kwe-20 kunye ne-35.
d.Ukulungiswa kweNkqubo: Nceda ulungiselele kwitafile yokusebenza ecocekileyo ye-ultra, kunye ne-pipettor kunye ne-reaction tube ngaphandle kwe-nuclease residu;kucetyiswa ukuba usebenzise intloko yompu kunye nesihluzi.Kuphephe ungcoliseko olunqamlezayo kunye nokungcoliseka kwe-aerosol.
2. UkuQhubela ibhayisekileUmgaqo-nkqubo
| Umjikelo inyathelo | Temp. | Ixesha | Imijikelo |
| Reverse transcription | 50℃a | 20mins | 1 |
| I-denaturation yokuqala | 95℃ | 5 imiz | 1 |
| Ukusabela kokukhulisa | 95℃ | 15sec |
40-45 |
| 60℃ b | 30sec c |
Amanqaku:
a.Reverse transcription:Iqondo lobushushu lingakhetha 42°C okanye 50°C.
b.Ukusabela kwe-Amplification: Ubushushu buhlengahlengiswa ngokwexabiso le-Tm lee-primers eziyilelweyo.
c.Ukufunyanwa komqondiso we-Fluorescence:Nceda usete inkqubo yovavanyo ngokweemfuno zesixhobo sokufunda.
Amanqaku
Nceda unxibe iPPE eyimfuneko, efana nedyasi yelebhu kunye neeglavu, ukuqinisekisa impilo nokhuseleko lwakho.
