2×Sensi Direct Premix-UNG (Probe qPCR)
Cat Nama: HCB5151A
I hangaia te SensiDirect Premix-UNG (Probe qPCR) ki te mahi PCR tika mai i nga tauira kaore he tangohanga DNA me te whakarite tauira.Ko tenei reagent he DNA polymerase wera-timata, uracil DNA glycosylase (UNG), RNase Inhibitor, MgCl2, dNTPs (me te dUTP hei utu mo te dTTP), me nga kaiwhakamau, mo te PCR ine (qPCR).Ko tenei reagent e hanga ana i te aukati-kaha te aukati, na reira ka taea te whakamahi ki te kitea o nga tauira penei i te korokoro korokoro, te huware, te toto toto katoa, te plasma, me te serum kaore he tangohanga DNA.Ka whakamahia e te reagent te parepare rangatira mo te qPCR me nga whaamu whakauru o te polymerase DNA anti-inhibitory me te UNG enzyme.Na reira, ka taea e ia te whakakaha pai o nga ira whaanui i roto i nga tauira kei roto nga aukati me te aukati i te whakakaha pai teka na te toenga PCR me te parahanga aerosol.He hototahi tenei reagent ki te nuinga o nga taputapu PCR ine kareoro, penei i te Applied Biosystems, Eppendorf, Bio-Rad, Roche me etahi atu.
Waehanga
1. 50×SensiDirect Enzyme/UNG Mix
2. 2×SensiDirect Premix Buffer (dUTP)
Nga tikanga rokiroki
Ko nga waahanga katoa me pupuri ki te -20 ℃ mo te rokiroki mo te wa roa me te 4 ℃ mo te 3 marama.Whakaranu pai i muri i te whakamaroketanga me te centrifuge i mua i te whakamahi.A ape i te whakamaroke i nga wa katoa.
Kawa Paihikara
| Hipanga | Te pāmahana | Te wa | Porohita |
| Te nakunaku | 50 ℃ | 2min | 1 |
| Whakahohenga Polymerase | 95℃ | 1-5min | 1 |
| Denature | 95℃ | 10-20s | 40-50 |
| Whakawhanaunga/Torohanga | 56-64 ℃ | 20-60s |
Tohutohu Pipeting
| Reagent | Rōrahi ia tauhohenga | Rōrahi ia tauhohenga | Te Whakamutunga Whakamutunga |
| 2×SensiDirect Premix Buffer (dUTP) | 12.5µL | 25µL | 1× |
| 50×SensiDirect Enzyme/UNG Mix | 0.5µL | 1µL | 1× |
| 25×Primer-Probe Mix1, 2 | 1µL | 2µL | 1× |
| Tauira3, 4 | - | - | - |
| ddH2O | - | - | - |
| Rōrahi katoa | 25 μL | 50 μL | - |
1. Ko te nuinga o te 0.2μM te kukū whakamutunga o te tuatahi.Mo nga hua pai ake, ka taea te arotau te kukū tuatahi i roto i te awhe o te 0.2-1μM.
2. Ko te tikanga, ka taea te arotau te kukū rangahau i roto i te awhe o te 0.1-0.3μM.Ko te kukū tino pai o te tirotiro e pa ana ki te taputapu whakakaha PCR i te waa tuuturu, te momo tirotiro, me te momo matū tapanga rewharewha.Titiro koa ki te pukapuka taputapu, ki nga whakaritenga motuhake ranei o ia rangahau kareoro.
3. Kei roto i nga momo tauira rereke nga momo momo me nga ihirangi o te aukati me te tau kape o te ira e whai ana.Ko te rahinga tauira me whakaarohia ma te ahuatanga pono.Hangaia he waimehatanga o te tauira ma te taapiri i te wai kore-karihi, TE Buffer ranei, ki te tika.
4. Rarahi o nga tauira rereke e taunaki ana:
| Tauira | Rōrahi mō te kotahi 50 μL tauhohenga | Mōrahi ōwehenga |
| Te toto toto katoa | 2.5 μL | 5% |
| Plasma | 15 μL | 30% |
| Serum | 10 μL | 20% |
| Horokoro korokoro | 10 μL | 20% |
| Huware | 10 μL | 20% |
Mana Kounga
1. Te kitenga mahi: te tairongo, te motuhake me te tukurua o te qPCR.
2. Karekau he mahi karihi o waho: kaore he endonuclease me te parahanga exonuclease.
Tuhinga o Hua
1. Ka whakamahia e tenei hua he momo hou o te DNA polymerase wera-tīmatanga, ka taea te whakahohe i roto i te 1-5 meneti. I te mea kua arotauhia tana parepare tauhohenga, he pai ake mo te PCR ine ine rewharewha maha ranei ma te whakamahi tikanga tirotiro.
2. Mēnā he iti rawa te uara Rn o te whakakaha PCR, kei te tino arai ranei te whakakaha, ka heke te rahi o te tauira, te whakanui ake i te rōrahi tauhohenga, te waimehatanga o mua ranei o te tauira ka pai ake nga hua.
3. Ko te kohinga o te toto, o te huware, o te mimi, o te korokoro, me te aha, me whai i nga whakaritenga paearu haumanu, ka taea te whakamahi tauira hou hei aukati i te paheketanga o te waikawa karihi.
4. I te mea he rereke nga amplicons rereke ki te whakamahi i te dUTP me te aro ki te UNG, me arotau nga reagents mena ka heke te arokitenga i te wa e whakamahi ana i te punaha UNG.Tena koa whakapiri mai ki a maatau mo te tautoko hangarau mena ka hiahiatia.
5. Hei karo i te whakakaha o nga hua PCR kawe i waenga i nga tauhohenga kotahi-taahiraa, me hiahia te waahi whakamatautau me te pipette mo te whakakaha.Me mahi me nga karapu me te huri pinepine me te kore e whakatuwhera i te ngongo tauhohenga i muri i te whakakaha PCR.
