Multiplex qPCR Probe Premix
Nambala ya mphaka: HCB5051A
TaqMan multiplex qPCR Master Mix (Dye Based) ndi yankho lachidule la 2 × zenizeni zenizeni zokulirapo za PCR zodziwika ndi kukhudzika kwakukulu komanso kutsimikizika, komwe kumakhala kobiriwira, ndipo kumakhala ndi zotsatira za kuwonjezera zitsanzo.Izi ndi 2 × Mix pre-mixed reagent yomwe imathandizira mpaka zinayi za fulorosenti zochulukira PCR munjira imodzi bwino.Chogulitsachi chimakhala ndi njira yosinthira ma genetic kuti ayambitse Taq enzyme, kumathandizira kwambiri kukhudzika kwachidziwitso komanso kutsimikizika.Nthawi yomweyo, mankhwalawa amathandizira kwambiri chotchinga chamitundu yambiri, chomwe chingathe kupititsa patsogolo kukulitsa kwa zomwe zimachitika ndikulimbikitsa kukulitsa bwino kwa ma tempuleti otsika kwambiri.Izi zitha kugwiritsidwa ntchito posanthula ma genotyping ndi ma multiplex.
Kufotokozera
| Hot Start | Yoyambira yotentha yomangidwa |
| Njira yodziwira | Kuzindikira koyambirira |
| Njira ya PCR | qPCR |
| Polymerase | Taq DNA polymerase |
| Mtundu wa zitsanzo | DNA |
Zosungirako
Zogulitsazo zimatumizidwa ndi ayezi wouma ndipo zimatha kusungidwa ku -25 ~ -15 ℃ kwa 2 chaka.
Malangizo
1. Zimene anachitaDongosolo
| Zigawo | Kuchuluka (μL) | Kukhazikika Kwambiri |
| 2 × TaqMan multiplex qPCR Master Mix | 12.5 | 1 × pa |
| Kusakaniza koyambirira (10 μmol/L) a | × | 0.1 - 0.5 μmol / L |
| Kusakaniza kwa probe (10 μmol/L)b | × | 50 - 250 nmol / L |
| Mtundu wa Rox | 0.5 | 1 × pa |
| DNA / cDNA template | 1-10 | - |
| ddH2O | mpaka 25 | - |
Ndemanga:Sakanizani bwino musanagwiritse ntchito kuti mupewe kugwedezeka kwakukulu.
a.Kuphatikizika koyambira: Kusakaniza koyambira kumakhala ndi mapeyala angapo a zoyambira, nthawi zambiri zoyambira zilizonse pamlingo womaliza wa 0.2 μmol/L ndipo zimathanso kusinthidwa pakati pa 0.1 ndi 0.5 μmol/L moyenerera.
b.Kukhazikika kwa probe: Probe Mix ili ndi ma probe angapo okhala ndi ma siginecha osiyanasiyana a fulorosenti, ndipo kuchuluka kwa kafukufuku uliwonse kumatha kusinthidwa pakati pa 50 ndi 250 nmol/L malinga ndi momwe zinthu zilili.
1.Rox dye reference: Imagwiritsidwa ntchito pa Real Time PCR chida chokulirapo monga Applied Biosystems kukonza cholakwika cha siginecha ya fluorescence yopangidwa pakati pa zitsime;mankhwalawa alibe Rox dye reference.Cas#10200 imalimbikitsidwa ngati ikufunika.
2.Template dilution: qPCR imakhudzidwa kwambiri, ndipo tikulimbikitsidwa kuti muchepetse template kuti mugwiritse ntchito.Ngati template ndi cDNA stock solution, voliyumu ya template siyenera kupitirira 1/10 ya voliyumu yonse.
3.Dongosolo lamachitidwe: 25μL, 30μL kapena 50 μL akulimbikitsidwa kuti atsimikizire kuchita bwino komanso kubwereza kwa kukulitsa kwa jini.
4.Kukonzekera kwadongosolo: Chonde konzekerani mu benchi yoyera kwambiri, ndipo gwiritsani ntchito nsonga ndi machubu ochitira popanda zotsalira za nyukiliya;tikulimbikitsidwa kugwiritsa ntchito nsonga ndi makatiriji fyuluta.Pewani kuipitsidwa ndi aerosol ndi kuipitsidwa.
2.Pulogalamu yamachitidwe
| Sitepe yozungulira | Temp. | Nthawi | Zozungulira |
| Chidziwitso choyambirira | 95 ℃ | 5 mins | 1 |
| Denaturation | 95 ℃ | 15mphindi | 45 |
| Zowonjezera / Zowonjezera | 60 ℃ | 30mphindi |
Ndemanga:
1.Annealing / Extension: Kutentha ndi nthawi zitha kusinthidwa moyenera malinga ndi mtengo woyambira wa Tm.
2.Kupeza siginecha ya Fluorescence: Nthawi yopeza siginecha ya fluorescence yofunikira pazida zosiyanasiyana zozindikirira qPCR ndi yosiyana, chonde ikani molingana ndi nthawi yochepa.Nthawi ya zida zingapo zodziwika bwino imayikidwa motere:
20 sec: Applied Biosystems 7700, 7900HT, 7500 Fast
31 sec: Applied Biosystems 7300
32 sec: Applied Biosystems 7500
Zolemba
Chonde valani PPE yofunikira, malaya a labu ndi magolovesi, kuti mutsimikizire thanzi lanu ndi chitetezo!
