2×PCR Master Mix (e aunoa ma se vali)
O le PCR Master Mix o se ituaiga o vaifofo masani PCR premixed lea ua sauni e fa'aoga, e aofia ai le Taq DNA Polymerase, dNTP mix MgCl2 ma le fa'alelei lelei.I le taimi o le tali, naʻo le faʻaulu ma le faʻataʻitaʻiga e mafai ona faʻaopoopo mo le faʻalauteleina, lea e matua faafaigofieina ai laasaga o le faʻataʻitaʻiga.O lenei oloa o loʻo iai ni faʻamautu lelei ma e mafai ona teuina mo le 3 masina ile 4℃.O le oloa PCR e 3′-dA fa'aoso ma e faigofie ona fa'asolo ile T vector.
Tulaga Teuteu
E tatau ona teuina le oloa i le -25 ℃ ~ -15 ℃ mo le lua tausaga.
Fa'amatalaga
| Fa'amaoni (vs.Taq) | 1× |
| Amata vevela | No |
| Fa'atautau | 3′-A |
| Polymerase | Taq DNA Polymerase |
| Fa'asologa o Tali | SuperMix po'o Master Mix |
| Saosaoa Tali | Tulaga |
| Ituaiga Oloa | PCR Master Mix (2×) |
Faatonuga
1.Reaction System
| Vaega | Tele (μL) |
| DNA mamanu | Talafeagai |
| Muamua 1 (10 μmol/L) | 2 |
| Muamua 2 (10 μmol/L) | 2 |
| PCR Master Mix | 25 |
| ddH2O | I le 50 |
2.Polokalama Fa'aliga
| Laasaga taamilo | Vevela (°C) | Taimi | Ta'amilosaga |
| mua'i denaturation | 94 ℃ | 5min | 1 |
| Denaturation | 94 ℃ | 30 sec | 35 |
| Fa'asusu | 50-60 ℃ | 30 sec | |
| Fa'aopoopoga | 72 ℃ | 30-60sec/kb | |
| Fa'aopoopo Mulimuli | 72 ℃ | 10min | 1 |
Fa'amatalaga:
1) Fa'aoga fa'ata'ita'iga: 50-200 ng genomic DNA;0.1-10 ng plasmid DNA.
2) Mg2+fa'atonuga: O lenei oloa e aofia ai le 3 mM o MgCl2 talafeagai mo le tele o tali PCR.
3) Su'ega vevela: Fa'amolemole va'ai i le fa'atatau Tm tau o Primers.E mafai ona seti le vevela o le annealing i le 2-5 ℃ maualalo ifo nai lo le taua o le faʻamuamua.
4) Taimi fa'aopoopo: Mo fa'amatalaga mole, 30 sec/kb e fautuaina.Mo gene cloning, 60sec/kb ua fautuaina.
Fa'amatalaga
1.Mo lou saogalemu ma lou soifua maloloina, fa'amolemole fai ni ofu talaloa ma totigilima fa'aaoga mo le taotoga.
2.Mo suʻesuʻega faʻaaoga naʻo!
