2×PCR Master Mix (me ka waiʻole)
ʻO ka PCR Master Mix kahi ʻano o ka PCR premixed solution maʻamau i mākaukau e hoʻohana, me ka Taq DNA Polymerase, dNTP mix MgCl2 a me ka buffer optimized.I ka wā o ka pane ʻana, hiki ke hoʻohui ʻia ka primer a me ka template no ka hoʻonui ʻana, ka mea e maʻalahi loa i nā ʻanuʻu hana o ka hoʻokolohua.Aia kēia huahana i nā stabilizers maikaʻi loa a hiki ke mālama ʻia no 3 mau mahina ma 4 ℃.He 3′-dA protrusion ka huahana PCR a hiki ke hoʻopaʻa maʻalahi i ka vector T.
Nā Kūlana Waihona
Pono e mālama ʻia ka huahana ma -25 ℃ ~ -15 ℃ no ʻelua mau makahiki.
Nā kikoʻī
| Pono (vs.Taq) | 1× |
| Hoomaka Wela | No |
| Ke kau ʻana | 3′-A |
| Polymerase | Taq DNA Polymerase |
| Hōʻike Manaʻo | SuperMix a i ʻole Master Mix |
| Ka wikiwiki o ka pane | Kūlana |
| ʻAno Huahana | Hui Pū ʻIa PCR (2×) |
Nā kuhikuhi
1.Pūnaehana Manaʻo
| Nā ʻāpana | Nui (μL) |
| DNA template | Kūpono |
| Kumuhana 1 (10 μmol/L) | 2 |
| Kumuhana 2 (10 μmol/L) | 2 |
| PCR Master Mix | 25 |
| ddH2O | I ka 50 |
2.Hoʻolaha Hoʻolaha
| Kaʻina kaʻapuni | Mahana (°C) | Manawa | Nā pōʻaiapuni |
| Pre-denaturation | 94 ℃ | 5 min | 1 |
| Denaturation | 94 ℃ | 30 sec | 35 |
| Hoʻopili | 50-60 ℃ | 30 sec | |
| Hoʻonui | 72 ℃ | 30-60sec/kb | |
| Hoʻonui hope | 72 ℃ | 10 min | 1 |
Nā memo:
1) Ka hoʻohana ʻana i ka laʻana: 50-200 ng genomic DNA;0.1- 10 ng plasmid DNA.
2) Mg2+ka manaʻo: Loaʻa i kēia huahana he 3 mM o MgCl2 kūpono no ka hapa nui o nā hopena PCR.
3) Ke ana wela: E ʻoluʻolu e nānā i ka waiwai Tm kumu o nā Primers.Hiki ke hoʻonohonoho ʻia ka mahana annealing i 2-5 ℃ haʻahaʻa ma mua o ka waiwai kumu o ka primer.
4) Ka manawa hoʻonui: No ka ʻike molekala, ʻōlelo ʻia ʻo 30 sec/kb.No ka hoʻopalekana gene, 60sec/kb ua paipai ʻia.
Nā memo
1.No kou palekana a me kou olakino, e ʻoluʻolu e ʻaʻahu i nā ʻaʻahu lab a me nā mīkina lima hoʻopau no ka hana.
2.No ka noiʻi wale nō!
