2 × Byihuta bya Taq Bivanze
Injangwe No: HCR2016A
2 Rap Rapid Taq Super Mix ishingiye kuri Taq DNA Polymerase yahinduwe, ikongeramo ibintu bikomeye byo kwagura, ibintu byongera imbaraga hamwe na sisitemu ya buffer nziza, hamwe nubushobozi buhanitse bwo kongera imbaraga.Amplification yihuta yibishusho byoroshye nka genome muri 3 kb igera kuri 1-3 sec / kb, naho iy'inyandikorugero yoroshye nka plasmide muri 5 kb igera kuri 1 sec / kb.Ibicuruzwa birashobora kubika cyane igihe cya PCR.Mugihe kimwe, kuvanga birimo dNTP na Mg2 +, bishobora kongerwaho gusa wongeyeho primers na templates, nabyo byoroshya cyane intambwe yimikorere yubushakashatsi.Byongeye kandi, kuvanga birimo irangi ryerekana amashanyarazi, rishobora kuba electrophorei nyuma yo kwitwara.Ibikoresho birinda iki gicuruzwa bituma kuvanga bikomeza ibikorwa bihamye nyuma yo gukonjeshwa no gukonja.Itsinda rya 3'-iherezo A ryibicuruzwa bya PCR birashobora gukoronizwa byoroshye muri T vector.
Ibigize
2 × Byihuta bya Taq Bivanze
Ububiko
PCR Master Mix ibicuruzwa bigomba kubikwa kuri -25 ~ -15 ℃ kumyaka 2.
Ibisobanuro
| Ibisobanuro ku bicuruzwa | Byihuta Taq Bivanze |
| Kwibanda | 2 × |
| Intangiriro ishyushye | Byubatswe muri Hot Itangira |
| Kurenga | 3′-A |
| Umuvuduko wo kwitwara | Byihuta |
| Ingano (Igicuruzwa cyanyuma) | Kugera kuri 15 kb |
| Ibisabwa mu bwikorezi | Urubura rwumye |
Amabwiriza
1. Sisitemu yo Kwitwara (50 μL)
| Ibigize | Ingano (μL) |
| Icyitegererezo ADN * | bikwiye |
| Imbere ya primer (10 μ mol / L) | 2.5 |
| Subiza primer (10 μ mol / L） | 2.5 |
| 2 × Byihuta bya Taq Bivanze | 25 |
| ddH2O | kugeza kuri 50 |
2.Amasezerano yo kongera imbaraga
| Intambwe zizunguruka | Ubushyuhe (° C) | Igihe | Amagare |
| Kubanziriza | 94 | Imin | 1 |
| Gutandukana | 94 | Amasegonda 10 |
28-35 |
| Annealing | 60 | 20 amasegonda | |
| Kwagura | 72 | 1-10 amasegonda / kb |
Basabwe gukoresha inyandikorugero zitandukanye:
| Ubwoko bw'icyitegererezo | Urwego rwo gukoresha igice (sisitemu ya 50 μL reaction) |
| ADN ya ADN cyangwa E. coli y'amazi | 10-1,000 ng |
| ADN ya plasma cyangwa virusi | 0.5-50 ng |
| cDNA | 1-5 µL (ntibirenze 1/10 cyubunini bwa reaction ya PCR) |
| Basabwe gukoresha inyandikorugero zitandukanye | |
Inyandiko:
1.Koresha reagent: gukonjesha rwose no kuvanga mbere yo gukoresha.
2. Ubushyuhe bwa Annealing: Ubushyuhe bwa annealing nigiciro rusange cya Tm, kandi gishobora no gushyirwaho 1-2 ℃ munsi ugereranije na primer Tm.
3. Umuvuduko wo kwaguka: Shiraho 1 sec / kb kubishusho byoroshye nka genome na E. coli muri 1 kb;shiraho 3 sec / kb kubishusho byoroshye nka 1-3 kb genome na E. coli;shiraho amasegonda 10 / kb kubishusho bigoye hejuru ya 3 kb genome na E. coli.Urashobora gushiraho agaciro kuri 1 sec / kb kubishusho byoroshye nka plasmid iri munsi ya 5 kb, 5 sec / kb kubishusho byoroshye nka plasmid iri hagati ya 5 na 10 kb, na 10 sec / kb kubishusho byoroshye nka plasmide irenze 10 kb.
Inyandiko
1. Kubwumutekano wawe nubuzima bwawe, nyamuneka wambare amakoti ya laboratoire hamwe na gants imwe ikoreshwa.
2. Iki gicuruzwa nikoreshwa mubushakashatsi GUSA!
