2 × PCR Super Mix (hamwe n'irangi)
2 × PCR Master mix irimo Taq DNA Polymerase, dNTPs, nibindi bikoresho bisabwa PCR.Master mix ihamye amezi 3 kuri 4 ℃ hamwe na stabilisateur yacu yihariye.Mbere yo kuvanga igisubizo cyateguwe neza kuri PCR isanzwe kandi yiteguye gukoresha wongeyeho inyandikorugero ya ADN na primers.Ibicuruzwa bya PCR birashobora gupakirwa muburyo bwa electrophoreis hamwe na bromophenol yabanje kwishyiriraho irangi ry'ubururu.Ibicuruzwa byongerewe imbaraga birimo 3 '-dA protrusion kandi birashobora gukoronizwa byoroshye muri T vector.2 × PCR Master mix yoroshya inzira ya PCR kandi igabanya kwanduza.
Ububiko
Ibicuruzwa bigomba kubikwa kuri -25 ℃ ~ -15 ℃ kumyaka 2.
Ibisobanuro
| Ubudahemuka (vs.Taq) | 1 × |
| Intangiriro ishyushye | No |
| Kurenga | 3 '-A |
| Polymerase | Taq ADN Polymerase |
| Imiterere | SuperMix cyangwa Master mix |
| Umuvuduko wo Kwitabira | Bisanzwe |
| Ubwoko bwibicuruzwa | PCR Master mix (2x) |
Amabwiriza
1.Sisitemu yo Kwitabira
| Ibigize | Umubumbe(μL) |
| Icyitegererezo ADN | Birakwiriye |
| Primer 1 (10 μ mol / L) | 2 |
| Primer 2 (10 μ mol / L） | 2 |
| 2 × PCR Master mix | 25 |
| ddH2O | kugeza kuri 50 |
2.Amasezerano yo kongera imbaraga
| Intambwe zizunguruka | Ubushyuhe (° C) | Igihe | Amagare |
| Gutandukana kwambere | 94 | Imin | 1 |
| Gutandukana | 94 | 30 amasegonda | 35 |
| Annealing | 50-60 | 30 amasegonda | |
| Kwagura | 72 | 30-60 amasegonda / kb | |
| Kwagura kwa nyuma | 72 | Imin. 10 | 1 |
Icyitonderwa:
1) Ikoreshwa ry'icyitegererezo: ADN genomic 50-200ng;0.1-10ng ADN ya plasmid.
2) Mg2+kwibanda: Iki gicuruzwa kirimo 3mM ya MgCl2, ikwiranye na PCR nyinshi.
3) Ubushyuhe bwa Annealing: Nyamuneka reba kuri Tm theoretical Tm agaciro ka primers.Ubushuhe bwa annealing burashobora gushirwa kuri 2-5 ℃ munsi kurenza agaciro kamahame ya primer.
4) Igihe cyo kwaguka: Kubiranga molekuline, birasabwa 30 sec / kb.Kubijyanye na gene, 60 sec / kb birasabwa.
Inyandiko
1.Ibicuruzwa bya PCR hamwe na 2 × PCR Master mix ntibikwiye kuri polyacrylamide gel electrophorei.
2.Kubwumutekano wawe nubuzima bwawe, nyamuneka wambare amakoti ya laboratoire hamwe na gants imwe ikoreshwa.
3.Ikoreshwa mubushakashatsi ikoreshwa GUSA!
