5×Neoscript Tere RT-qPCR Premix me-UNG
Cat Nama: HCB5142A
Ko te Neoscript Fast RT Premix-UNG (Probe qRT-PCR) he ranunga e tika ana mo te whakamaarama whakamuri kotahi-taahiraa me te PCR ine (qRT-PCR).Ka tautokohia i mua i te whakakotahitanga o nga mea tuatahi me nga rangahau ka noho pumau i muri i te wa roa o te rokiroki i te pāmahana iti.Ko te tauira ka whakamatauria ka taea te taapiri tika i te wa e whakamahi ana, me te kore he mahi whakatuwhera ngongo/pipetting.Ka whakaratohia e tenei hua nga waahanga, hei tauira, te wera-tīmatanga DNA polymerase, M-MLV, te wera-kore uracil DNA glycosylase (TS-UNG), RNase Inhibitor, MgCl2, dNTPs (me te dUTP hei utu mo te dTTP), me te whakapumau.Ma te whakahiatotanga tere o te whakawhitinga whakamuri me te DNA polymerase, ka taea te whakaoti i te whakakaha PCR i roto i te 20-40 meneti.Ka whakamahia e tenei reagent he parepare motuhake mo te qPCR me nga whaamu whakauru o te whaariki whakakaha anti-inhibitory me te UNG enzyme.Na reira, ka taea e ia te whakakaha pai o nga ira e whai ana me te aukati i te whakakaha teka na te toenga PCR me te parahanga aerosol.He hototahi tenei reagent ki te nuinga o nga taputapu PCR ine ine rerehua mai i nga kaihanga penei i te Applied Biosystems, Eppendorf, Bio-Rad me Roche.
Waehanga
1.25×Neoscript Tere RTase/UNG Mix
2.5×Neoscript Tere RT 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.
qRT-PCR Reaction System Whakaritenga
| Waehanga | 25μLPūnaha | 50μLPūnaha | Te Whakamutunga Whakamutunga |
| 5×Neoscript Tere RT Premix Buffer (dUTP) | 5μL | 10μL | 1× |
| 25×Neoscript Tere RTase/UNG Mix | 1μL | 2μL | 1× |
| 25×Primer-Probe Mixa | 1μL | 2μL | 1× |
| RNA tauirab | – | – | – |
| ddH2O | Tae atu ki te 25μL | Tae atu ki te 50μL | – |
1) a.Ko te kukū whakamutunga o te tuatahi ko te nuinga o te 0.2μM.Mo nga hua pai ake, ka taea te arotau te kukū tuatahi i roto i te awhe o te 0.2-1μM.Ko te tikanga, ka taea te arotau te kukū o te rangahau i roto i te awhe o te 0.1-0.3μM.
2) b.I te wa e whakamahi ana i te Tikanga PCR tere, ko te whakanui ake i te kukū o nga papa tuatahi me nga rangahau ka pai ake nga hua whakakaha ake, me te pai ake o te owehenga.
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 waimeha o te tauira ki te wai kore karihi, TE Buffer ranei, ki te tika.
Tauhohenga Coati
| Tikanga PCR auau | Te Tikanga PCR Tere | ||||||
| Tikanga | Temp. | Te wa | Porohita | Tikanga | Temp. | Te wa | Porohita |
| Whakamuri Whakamuri | 50 ℃ | 10-20 meneti | 1 | Whakamuri Whakamuri | 50 ℃ | 5min | 1 |
| Polymerase Whakahohenga | 95℃ | 1-5 meneti | 1 | Polymerase Whakahohenga | 95℃ | 30s | 1 |
| Te whakahekenga | 95℃ | 10-20s | 40-50 | Te whakahekenga | 95℃ | 1-3s | 40-50 |
| Te whakaene a Toronga | 56-64 ℃ | 20-60s | Te whakaene a Toronga | 56-64 ℃ | 3-20s | ||
Mana Kounga
1.Te kitenga mahi: te tairongo, te motuhake me te tukurua o te qPCR.
2.Karekau he mahi karihi o waho: karekau he endonuclease me te parahanga exogenous.
Notes
1.Ko te tere whakakaha o te polymerase DNA tere he iti iho i te 1kb/10s.He rereke te whakamahana me te whakamahana o nga taputapu PCR rereke, nga tikanga whakahaere i te pāmahana me te kawe waiariki, na reira he mea nui te arotautanga o to kukū tuatahi/tirotiro me te tikanga whakahaere me to taputapu PCR tere.
2.Ko tenei hua e mahi ana i te whanuitanga o te whakamahinga, a he pai mo te tātari irapo tairongo teitei.Ko te tikanga PCR e toru-taahiraa e taunakitia ana mo nga kaimatai he iti te pāmahana whakaeneene, hei whakakaha ranei i nga kongakonga roa neke atu i te 200 bp.
3.I te mea he rereke nga amplicons he rereke te kaha o te whakamahi o te dUTP me te rerekee o te tairongo 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.
4.Hei karo i te whakakaha o nga hua PCR kawe, me whai waahi whakamatautau me te pipette mo te whakakaha.Me mahi me nga karapu me te huri pinepine, kaua hoki e whakatuwhera i te ngongo PCR i muri i te whakakaha.
