2 × PCR Super Mix (ya na agba)
2× PCR Master Mix nwere Taq DNA Polymerase, dNTPs, na ihe PCR ndị ọzọ achọrọ.Ngwakọta Master ahụ kwụsiri ike maka ọnwa 3 na 4 ℃ ya na ndị nrụpụta ahaziri iche.A na-emezi ihe ngwọta tupu ngwakọta maka PCR na-emekarị ma dị njikere iji site na ịgbakwunye ndebiri DNA na primers.Enwere ike ibunye ngwaahịa PCR ozugbo maka electrophoresis nwere agba anụnụ anụnụ bromophenol ebugoro ebugoro.Ngwaahịa ndị a na-amụbawanye nwere 3 '-dA protrusion na enwere ike itinye ya ngwa ngwa na vector T.Ngwakọta 2 × PCR Master Mix na-eme ka usoro PCR dị mfe ma belata mmetọ.
Ọnọdụ Nchekwa
Ngwaahịa kwesịrị ịchekwa na -25 ℃ ~ -15 ℃ maka 2 afọ.
Nkọwapụta
| Ikwesị ntụkwasị obi (vs.Taq) | 1× |
| Mmalite na-ekpo ọkụ | No |
| N'ofefe | 3'-A |
| Polymerase | DNA Polymerase |
| Ụdị mmeghachi omume | SuperMix ma ọ bụ Master Mix |
| Ọsọ mmeghachi omume | Ọkọlọtọ |
| Ụdị ngwaahịa | Ngwakọta PCR Master Mix (2x) |
Ntuziaka
1.Sistemụ mmeghachi omume
| Ngwa | Olu(μL) |
| DNA template | Kwesịrị ekwesị |
| Nke mbụ 1 (10 μmol/L) | 2 |
| Nke mbụ 2 (10 μmol/L) | 2 |
| 2 × PCR Master Mix | 25 |
| ddH2O | ruo 50 |
2.Protocol Amplification
| Usoro okirikiri | Okpomọkụ (°C) | Oge | okirikiri |
| denaturation nke mbụ | 94 | 5 min | 1 |
| Denaturation | 94 | 30 sk | 35 |
| Na-ewe iwe | 50-60 | 30 sk | |
| Mgbatị | 72 | 30-60 sk/kb | |
| Mgbatị ikpeazụ | 72 | 10 min | 1 |
Mara:
1) Ojiji ihe atụ: 50-200ng DNA genomic;0.1-10ng DNA plasmid.
2) mg2+itinye uche: Ngwaahịa a nwere 3mM nke MgCl2, dabara maka ọtụtụ mmeghachi omume PCR.
3) Ọkụ na-ekpo ọkụ: Biko rụtụ aka na uru Tm nke primers.Enwere ike ịtọ okpomọkụ na-ekpo ọkụ ka ọ bụrụ 2-5 ℃ dị ala karịa uru ụkpụrụ nke primer.
4) Ogologo oge: Maka njirimara molecular, 30 sec / kb ka akwadoro.Maka mmepụta mkpụrụ ndụ ihe nketa, 60 sk/kb ka akwadoro.
Ihe ndetu
1.Ngwaahịa PCR nwere 2 × PCR Master Mix adịghị adabara polyacrylamide gel electrophoresis.
2.Maka nchekwa na ahụike gị, biko yiri uwe lab na gloves enwere ike iji rụọ ọrụ.
3.A na-eji ya maka nyocha naanị!
