Robusstart Taq DNA Polymerase
Ko te Robusstart Taq DNA Polymerase he DNA polymerase timatanga wera.Kaore e taea e tenei hua te aukati noa i te tauhohenga kore-motuhake i puta mai i te kore-motuhake o nga mea tuatahi, i te whakahiato tuatahi ranei i roto i te tukanga o te whakarite me te whakanui i te punaha PCR.Na reira, he tino pai, he pai ake mo te whakakaha i nga tauira kukū iti, a he pai mo te tauhohenga whakakaha PCR maha.I tua atu, he tino pai te whakamahi o tenei hua, a ka taea te whiwhi hua whakakaha i roto i nga momo tauhohenga PCR.
Waehanga
1.5 U/μL Robustart Taq DNA polymerase
2.10 × PCR Buffer II (Mg²+ kore utu) (kōwhiringa)
3.25 mM MgCl2(kōwhiringa)
* 10 × PCR Buffer II (Mg²+ kore utu) karekau he dNTP me Mg²+, tēnā tāpirihia ngā dNTP me te MgCl2i te wa e whakarite ana i te punaha tauhohenga.
Nga tono e manakohia ana
1.Whakanuia tere.
2.Whakanuia maha.
3.Ko te whakakaha tika o te toto, te pania, me etahi atu tauira.
4.Te kitenga mate manawa.
Tikanga Rokiroki
-20°C mo te rokiroki mo te wa roa, me uru pai i mua i te whakamahi, karohia te whakamao-kore-roa.
*Mena ka puta te ua i muri i te whakamatao, he mea noa;e taunaki ana kia taurite ki te pāmahana rūma i mua i te whakaranu me te whakamahi.
Whakamaramatanga Waeine
Ko tetahi waeine kaha (U) kua tautuhia ko te nui o te whaariki e whakauru ana i te 10 nmol o te deoxyribonucleotide ki roto i te waikawa-kore-waikawa i te 74°C mo te 30meneti ma te whakamahi i te DNA sperm sperm hohe hei tauira/primer.
Mana Kounga
1.SDS-PAGE electrophoretic purenga nui ake i te 98%.
2.Te tairongo whakakaha, te mana o te puranga-ki-te-puranga, te pumau.
3.Karekau he mahi karihi o waho, karekau he endonuclease o waho, he poke exonuclease ranei
Tohutohu
Tauhohenga Tatūnga
| Waehanga | Rōrahi (μL) | Te Whakamutunga Whakamutunga |
| 10 × PCR Buffer II (Mg²+ kore utu)a | 5 | 1× |
| dNTP (10mM ia dNTP) | 1 | 200 μM |
| 25 mM MgCl2 | 2-8 | 1-4 mM |
| Robusstart Taq DNA Polymerase (5U/μL) | 0.25-0.5 | 1.25-2.5 U |
| 25 × te ranunga tuatahib | 2 | 1× |
| tauira | - | ＜ 1 μg/hohenga |
| ddH2O | Ki te 50 | - |
Tuhipoka:
1) a.Karekau he dNTP me Mg²+ i roto i te parepare, me tapiri nga dNTP me MgCl2i te wa e whakarite ana i te punaha tauhohenga.
2) b.Mēnā ka whakamahia mō te qPCR/qRT-PCR, me tāpirihia ngā rangahau kōaro ki te pūnaha tauhohenga.Ko te tikanga, ko te kukū tuatahi o te 0.2 μM ka puta he hua pai;ki te he rawakore te mahi tauhohenga, ka taea te whakarite te kukū tuatahi i roto i te whānuitanga o 0.2-1 μM.Kei te nuinga papaitia te kukū rangahau i roto i te whānuitanga o 0.1-0.3 μM.Ka taea te whakahaere i nga whakamatautau rōnaki ki te kimi i te huinga pai o te tuatahi me te rangahau.
Kawa paihikara wera
| PCR auautukanga | |||
| Hipanga | Te pāmahana | Te wa | Nga huringa |
| Tuhinga o mua | 95℃ | 1-5 min | 1 |
| Te whakahekenga | 95℃ | 10-20 hēkona | 40-50 |
| Whakawhana / Toronga | 56-64 ℃ | 20-60 hēkona | |
| PCR teretukanga | |||
| Hipanga | Te pāmahana | Te wa | Nga huringa |
| Tuhinga o mua | 95℃ | 30 hēkona | 1 |
| Te whakahekenga | 95℃ | 1-5 hēkona | 40-45 |
| Whakawhana / Toronga | 56-64 ℃ | 5-20 hēkona | |
Notes
1.Ko te tere whakakaha o te DNA polymerase tere kia kaua e iti iho i te 1 kb/10 s.He rerekee te pikinga o te pāmahana me te hekenga, te aratau whakahaere pāmahana me te pai o te kawe wera o nga taputapu PCR rereke, na reira ka tūtohu kia arotauhia nga tikanga tauhohenga tino pai mo te taputapu PCR tere.
2.He tino pai te urutau o te punaha, he nui ake te motuhake me te tairongo.
3.He pai mo te whakamahi hei reagents rapunga PCR tairongo teitei, ka taea te whakamahi i nga tauhohenga whakakaha PCR maha.
4.5′→3′ mahi polymerase, 5′→3′ mahi exonuclease;kahore 3′→5′ mahi exonuclease;kore mahi whakatika.
5.He pai mo te whakamatautau tohu me te ine o te PCR me te RT-PCR.
6.Ko te 3′ te mutunga o te hua PCR he A, ka taea te whakakoi tika ki te vector T.
7.Ko te tikanga e toru-taahiraa e taunakitia ana mo nga kaimatai he iti te mahana whakaeneene, mo te whakakaha ranei i nga kongakonga roa ake i te 200 bp.
