2×PCR Super Mix (me te Waikano)
2× PCR Master Mix kei roto Taq DNA Polymerase, dNTPs, me etahi atu waahanga e hiahiatia ana e te PCR.E u ana te Master Mix mo te 3 marama i te 4 ℃ me o maatau whakapumau.Ko te otinga i mua i te whakaranu kua whakatikahia mo te PCR tikanga me te reri ki te whakamahi ma te taapiri i te tauira DNA me nga mea tuatahi.Ko nga hua PCR ka taea te uta tika mo te electrophoresis me te tae puru bromophenol i mua i utaina.Kei roto i nga hua kua whakanuia te 3'-dA te tohotanga ka taea te whakakoe ki roto i te vector T.Ko te 2×PCR Master Mix he whakamaarama i te mahi PCR me te whakaiti i te poke.
Nga Tikanga Rokiroki
Me penapena nga hua ki te -25 ℃ ~ -15 ℃ mo te 2 tau.
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 | Whakaranu Matua PCR (2x) |
Tohutohu
1.Pūnaha Tauhohenga
| Waehanga | Rōrahi(μL) |
| DNA tauira | E tika ana |
| Tuatahi 1 (10 μmol/L) | 2 |
| Tuatahi 2 (10 μmol/L) | 2 |
| 2× PCR Master Mix | 25 |
| ddH2O | ki te 50 |
2.Kawa Whakanui
| Nga hikoi hurihanga | Paemahana (°C) | Te wa | Nga huringa |
| Te whakahekenga tuatahi | 94 | 5 min | 1 |
| Te whakahekenga | 94 | 30 hēkona | 35 |
| Te whakaene | 50-60 | 30 hēkona | |
| Toronga | 72 | 30-60 hēkona/kb | |
| Toronga whakamutunga | 72 | 10 min | 1 |
Tuhipoka:
1) Te whakamahi tauira: 50-200ng DNA genomic;0.1-10ng DNA plasmid.
2) Mg2+kukū: Kei roto i tenei hua te 3mM o MgCl2, he pai mo te nuinga o nga tauhohenga PCR.
3) Paemahana whakaene: Tena koa tirohia te uara Tm ariā o nga mea tuatahi.Ka taea te whakarite i te pāmahana whakaene ki te 2-5 ℃ iti iho i te uara o te tuatahi.
4) Te wa whakaroa: Mo te tautuhi irapoi, 30 hekona/kb e taunaki ana.Mo te whakamaarama ira, 60 hēkona/kb te tūtohu.
Notes
1.Ko nga hua PCR me te 2× PCR Master Mix kaore e pai mo te polyacrylamide gel electrophoresis.
2.Mo to haumaru me to hauora, mau koti taiwhanga me nga karapu tuku mo te mahi.
3.Ka whakamahia mo te rangahau ANAKE!
