Multiplex One Step RT-qPCR Premix-UNG
Nambala ya mphaka: HCB5145A
Multiplex One Step RT-qPCR Probe Kit (UDG Plus) ndi kuchuluka kwa PCR zida kutengera RNA monga template.Pakuyesaku, kubwerezabwereza komanso kuchuluka kwa PCR kunachitika mu chubu lomwelo, zomwe zidapangitsa kuti ntchito yoyeserera ikhale yosavuta ndikuchepetsa chiopsezo cha kuipitsidwa.Mu chida ichi, cDNA yoyamba ya chingwe idapangidwa bwino ndi Reverse Transcriptase yosamva kutentha ndikukulitsidwa mochulukira ndi HotStart Tag DNA Polymerase.Zidazi zimakhala ndi buffer yokhathamiritsa ya MP, kusakanikirana kwa michere, ndi zina zambiri. Njira ya buffer ili kale ndi Mg.2+ndi dNTP.Kuphatikiza apo, zinthu zomwe zingalepheretse kukulitsa kwa PCR kosatchulika ndikuwongolera magwiridwe antchito angapo a qPCR zimawonjezedwa, zomwe zitha kuwonetsetsa kuti kukulitsa bwino ndikukwaniritsa kangapo kakulidwe kambiri.Dongosolo la dUTP/UDG lidawonjezedwa kuti lipewe kuopsa kwa kuipitsidwa kwa aerosol.
Zigawo
1. Bafa
2. Kusakaniza kwa enzyme
Kufotokozera
| Hot Start | Yoyambira yotentha yomangidwa |
| Njira yodziwira | Kuzindikira koyambirira |
| Njira ya PCR | Gawo limodzi RT-qPCR |
| Polymerase | Taq DNA polymerase |
| Mtundu wa zitsanzo | DNA |
Zosungirako
Mankhwalawa amatumizidwa ndi ayezi wouma ndipo amatha kusungidwa ku -25 ~ -15 ℃ kwa chaka chimodzi.Iyenera kupewa pafupipafupikuzimitsa-thaw.Ndi bwino kupulumutsa padera.
Malangizo
1.Zomwe anachita Dongosolo
| Zigawo | Kuchuluka (μL) | Kukhazikika Kwambiri |
| 2 × MP Buffer | 12.5 | 1 × pa |
| Kusakaniza kwa Enzyme | 1 | - |
| Kusakaniza koyambira/Probe (2.5 μM) | 3 | 0.3μM |
| Chithunzi cha RNA | 1-10 | - |
| RNase Free H2O | ku 25 | - |
Ndemanga:
Onetsetsani kuti mwasakaniza bwino musanagwiritse ntchito, pewani thovu lochulukirapo chifukwa cha kugwedezeka kwamphamvu.
a.Kusakaniza koyambira: Kusakaniza koyambira kuphatikiza zoyambira zambiri, kutengera momwe zinthu zilili bwino pakati pa 0.l ndi 1.0μM.
b.Kusakanizidwa kwa probe: Kusakaniza kwa probe kuphatikiza ma multiplex probe cholembera kusiyana kwa fulorosenti, kutengera momwe zinthu zilili mulingo woyenera wa kafukufukuyu mwina pakati pa 0.05 ndi 0.5μM.
c.Template dilution: qPCR imakhudzidwa kwambiri ndipo tikulimbikitsidwa kuti muchepetse template.Mtengo wa Ct wowongolera ndi woyenera pakati pa 20 ndi 35.
d.Kukonzekera dongosolo: Chonde konzekerani mu tebulo kopitilira muyeso woyera ntchito, ndi pipettor ndi anachita chubu popanda nuclease zotsalira;Ndi bwino kugwiritsa ntchito mfuti mutu ndi fyuluta chinthu.Pewani kuipitsidwa ndi aerosol ndi kuipitsidwa.
2. Wokometsedwa PanjingaNdondomeko
| Kuzungulira sitepe | Temp. | Nthawi | Zozungulira |
| Sinthani zomasulira | 50 ℃a | 20 mins | 1 |
| Chidziwitso choyambirira | 95 ℃ | 5 mins | 1 |
| Amplification reaction | 95 ℃ | 15mphindi |
40-45 |
| 60 ℃ b | 30mphindi c |
Ndemanga:
a.Reverse transcript: Kutentha kumatha kusankha 42°C kapena 50°C.
b.Amplification reaction: Kutentha kumasinthidwa malinga ndi mtengo wa Tm wa zoyambira zopangidwa.
c.Kupeza chizindikiro cha Fluorescence:Chonde ikani njira yoyesera molingana ndi zofunikira za buku la zida.
Zolemba
Chonde valani PPE yofunikira, monga malaya a labu ndi magolovesi, kuti mutsimikizire thanzi lanu ndi chitetezo.
