Superstart qPCR Premix me-UNG
Cat Nama: HCB5071E
Ko te Superstart qPCR Premix me-UNG he reagent motuhake i hangaia mo nga tauhohenga kounga me te ine ine PCR i te wa ma te whakamahi i te rapunga-a-te rangahau, i hangaia mo nga mahi lyophilization.Kei roto he pūrākau whakaoho wera wera Hotstart Taq me (DG), kua hirihia tana mahi enzyme Taq ki te pāmahana rūma, he kaha te aukati i te whakakaha kore-motuhake i puta mai i te whakahiatotanga kore-motuhake, te hanga dimer tuatahi ranei i raro i nga ahuatanga o te pāmahana iti, na reira ka pai ake. te motuhake o te tauhohenga whakakaha.Ko tenei reagent e whakamahi ana i te putunga motuhake qPCR me te punaha anti-contamination UNG/dUTP ki te whakatutuki tere i te tiimata wera, te whakapai ake i te pai me te maarama o nga tauhohenga qPCR.Ka taea e ia te tiki i nga pihinga paerewa pai i roto i te whānuitanga o nga waahi ine me te mahi tika i te ine, me te aukati i te whakakaha pai teka na te toenga o nga hua PCR, te poke aerosol ranei.He hototahi tenei reagent ki te nuinga o nga taputapu PCR ine kareoro mai i nga kaihanga penei i te Applied Biosystems, Eppendorf, Bio- Rad me Roche me etahi atu, me te whakaatu pai i te ahua lyophilized.
Te hanganga reagent
1. 5×HotstartPremix me-UNG (Mg2+kore utu) (DG)
2. 250 mM MgCl2
3. 4×lyoprotectant (kōwhiringa)
Nga Tikanga Rokiroki
Te rokiroki mo te wa roa i te -20 ℃;ka taea te penapena ki te 4 ℃ mo te 3 marama.Whakaranu pai i mua i te whakamahi me tekarohia te tio me te whakamaroke.
Kawa Paihikara
| Tikanga | Temp. | Te wa | Porohita |
| Te nakunaku | 50 ℃ | 2 min | 1 |
| Whakahohenga Polymerase | 95℃ | 1~5 min | 1 |
| Denature | 95℃ | 10~20 s | 40-50 |
| Te Whakanui me te Whakaroa | 56~64 ℃ | 20~60 s | 40-50 |
qPCR Liquid Reaction Syte takakau
|
Te tito |
25µL Rōrahi |
50µL Rōrahi |
Te Whakamutunga Whakamutunga |
| 5×HotstartPremix me-UNG(Mg2+kore utu) (DG) | 5µL | 10µL | 1× |
| 250mM MgCl2 | 0.45µL | 0.9µL | 4.5 mM |
| 4×lyoprotectant1 | 6.25µL | 12.5µL | 1× |
| 25×Primer-Probe Mix2 | 1µL | 2µL | 1× |
| DNA tauira3 | —— | —— | —— |
| ddH2O | Ki te 25µL | Ki te 50µL | —— |
1. Ko te kukū whakamutunga o te 0.2μM mo nga tuatahi ka puta he hua pai;ina he ngoikore te mahi tauhohenga, whakatikahia te kukū tuatahi i roto i te awhe o te 0.2-1μM ina hiahiatia.I te nuinga o te wa ka arotauhia te kukū o te rangahau i roto i te awhe o te 0.1-0.3μM na roto i nga whakamatautau rōnaki ki te kimi huinga tino pai.
2. He rereke te tau kape o nga ira e mau ana i roto i nga momo tauira;mehemea e tika ana, ka taea te mahi waimehatanga rōnaki hei whakatau i te rahinga taapiri tauira tino pai.
3. Ka taea te lyophilized tenei punaha;ka whakamahi nga kaihoko i tenei punaha kaore he whakaritenga whakamaroke-tio, ka taea te taapiri 4xlyoprotectant; mena kei te hiahiatia nga hua whakamaroke-tio, i te wa e whakamanahia ana nga hua o te reagents wai, me taapiri te 4xlyoprotectant hei whakarite kia rite ki nga waahanga punaha lyophilized me nga paanga.
I te wa e whakamahia ana te punahad mo te whakamaroke-tio, whakarerihia te pūnaha as e whai ake nei:
| Te tito | 25µL Pūnaha Tauhohenga |
| 5 ×HotstartPremix me-UNG (Mg2+kore utu) (DG) | 5µL |
| 250mM MgCl2 | 0.45µL |
| 4×lyoprotectant | 6.25µL |
| 25×Primer-Probe Mix | 1µL |
| ddH2O | Ki te 18~20µL |
* Mena ka hiahiatia etahi atu punaha mo te whakamaroke-tio, me korero motuhake.
Tikanga Lyophilizationss
| Tikanga | Temp. | Te wa | Tikanga | Te pehanga |
| I mua i te mīti | 4℃ | 30 min | Puritia |
1 atm |
| -50 ℃ | 60 min | Te whakamatao | ||
| -50 ℃ | 180 min | Puritia | ||
| Te whakamaroke tuatahi | -30 ℃ | 60 min | Whakawera |
Korehau Mutunga |
| -30 ℃ | 70 min | Puritia | ||
| Te whakamaroke tuarua | 25℃ | 60 min | Whakawera |
Korehau Mutunga |
| 25℃ | 300 min | Puritia |
2. Ko te tukanga lyophilization i runga ake nei mo te tohutoro anake.He rereke nga tawhā o nga momo hua me nga whakamaroke whakamaroke rereke, na reira ka taea te whakarereke kia rite ki nga mea pono.tikanga i te wa e whakamahia ana.
3. He pai pea nga momo mahi lyophilization mo nga rahi o te puranga o te lyophilizednga hua, no reira me tika te whakamanatanga o nga whakamatautau ka whakamahia mo te whakaputa nui.
Tohutohu mo te whakamahi lyophilized paura
1. Pokahia te paura lyophilized;
2. Tāpirihia te tauira waikawa nucleic ki te paura lyophilized me te tāpiri wai ki te 25µL;
3. Whakaranu pai ma te centrifugation ka rere ki runga miihini.
Mana Kounga:
1. Te whakamatautau mahi: te tairongo, te motuhake, te whakahoki ano o te qPCR.
2. Karekau he ngohe karihi o waho, karekau he poke endo/exonuclease.
Mōhiohio Hangarau:
1. Ka whakamahia e te Superstart qPCR Premix plus-UNG he pūrākau wera-tīmata whākōkī e taea ai te tīmata wera tere i roto i te 1~5 meneti;na roto i te hanga parepare motuhake e tika ana mo nga tauhohenga PCR ine ine korakora.
2. He teitei ake te motuhake e tino whakapai ake ai i te tairongo o te rewharewha ine ine PCR tepe rapunga, te hanga i nga pihi whakahiatotanga, ka kitea te whakapai ake i te uara korekore ki nga tauira kukū iti, e tika ana hei reagents PCR ine ine.
3. Mo nga mea tuatahi he iti ake te pāmahana whakaeneene, he roa ake ranei i nga kongakonga 200bp, ka tūtohutia kia 3-taahiraa.
4. He rereke te kaha o te whakamahinga o te dUTP me te aro ki te UNG enzyme mo nga momo ira e whaaia ana, na, ki te maataki te whakamahi i te punaha UNG ki te whakaheke i te maaramatanga o te kitenga, me whakatika me te arotau te punaha tauhohenga.Mena ka hiahiatia he tautoko hangarau me waea atu ki ta maatau kamupene.
5. Whakamahia nga waahi whakatapua me nga paipa i mua i muri mai i muri i te whakanui, mau karapu i te wa e mahi ana, ka whakakapi i nga wa katoa;kaua e whakatuwheratia te ngongo tauhohenga i muri i te otinga PCR hei whakaiti i te poke o nga tauira e nga hua PCR.
