2×PCR Master Mix (kahore he Waikano)
Ko te PCR Master Mix he momo otinga PCR premixed tikanga kua reri ki te whakamahi, tae atu ki te Taq DNA Polymerase, dNTP ranu MgCl2 me te parapara pai.I te wa o te tauhohenga, ko te tuatahi me te tauira ka taea te taapiri mo te whakakaha ake, he tino whakamaarama i nga mahi o te whakamatautau.Kei roto i tenei hua nga whakapumau pai, ka taea te rongoa mo te 3 marama ki te 4 ℃.Ko te hua PCR he toronga 3′-dA ka taea te whakakoe ki roto i te vector T.
Nga Tikanga Rokiroki
Me penapena te hua ki te -25 ℃~ -15 ℃ mo nga tau e rua.
Whakatakotoranga
| Pono (vs.Taq) | 1× |
| Tīmata wera | No |
| Irirangi | 3′-A |
| Polymerase | Taq DNA Polymerase |
| Hōputu Tauhohenga | SuperMix ranei Master Mix |
| Tere Hohenga | Paerewa |
| Momo Hua | PCR Master Mix (2×) |
Tohutohu
1.Pūnaha Tauhohenga
| Waehanga | Rahi (μL) |
| DNA tauira | E tika ana |
| Tuatahi 1 (10 μmol/L) | 2 |
| Tuatahi 2 (10 μmol/L) | 2 |
| PCR Master Mix | 25 |
| ddH2O | Ki te 50 |
2.Kawa Whakanui
| Nga hikoi hurihanga | Paemahana (°C) | Te wa | Nga huringa |
| Tuhinga o mua | 94 ℃ | 5min | 1 |
| Te whakahekenga | 94 ℃ | 30 hēkona | 35 |
| Te whakaene | 50-60 ℃ | 30 hēkona | |
| Toronga | 72 ℃ | 30-60sec/kb | |
| Toronga Whakamutunga | 72 ℃ | 10 meneti | 1 |
Tuhipoka:
1) Te whakamahi tauira: 50-200 ng DNA genomic;0.1- 10 ng plasmid DNA.
2) Mg2+kukū: Kei roto i tenei hua te 3 mM o MgCl2 e tika ana mo te nuinga o nga tauhohenga PCR.
3) Paemahana whakaene: Tena tirohia te uara Tm ariā o Primers.Ka taea te whakarite i te pāmahana whakaene ki te 2-5 ℃ iti iho i te uara o te tuatahi.
4) Te wa toronga: Mo te tautuhi irapoi, 30 hekona/kb e taunaki ana.Mo te ira ira, 60hekona/kb e taunaki ana.
Notes
1.Mo to haumaru me to hauora, mau koti taiwhanga me nga karapu tuku mo te mahi.
2.Mo te whakamahi rangahau anake!
