2×PCR Super Mix (ma le Vai)
2× PCR Master Mix e iai le Taq DNA Polymerase, dNTPs, ma isi vaega e manaʻomia PCR.O le Master Mix e mautu mo le 3 masina ile 4℃ fa'atasi ai ma a tatou fa'amautu fa'apitoa.O le fofo muamua-faafefiloi ua sili ona lelei mo PCR masani ma sauni e faʻaoga e ala i le faʻaopoopoina o DNA mamanu ma primers.O oloa PCR e mafai ona uta sa'o mo electrophoresis fa'atasi ai ma le vali lanumoana bromophenol.O oloa fa'ateleina e iai le 3 '-dA fa'aoso ma e faigofie ona fa'asolo ile T vector.O le 2×PCR Master Mix e fa'afaigofieina le faiga o le PCR ma fa'aitiitia ai le fa'aleagaina.
Tulaga Teuteu
O oloa e tatau ona teuina ile -25℃~-15℃ mo le 2 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 (2x) |
Faatonuga
1.Reaction System
| Vaega | Volume(μL) |
| DNA mamanu | Talafeagai |
| Muamua 1 (10 μmol/L) | 2 |
| Muamua 2 (10 μmol/L) | 2 |
| 2× PCR Master Mix | 25 |
| ddH2O | i le 50 |
2.Polokalama Fa'aliga
| Laasaga taamilo | Vevela (°C) | Taimi | Ta'amilosaga |
| Uluai denaturation | 94 | 5 minu | 1 |
| Denaturation | 94 | 30 sec | 35 |
| Fa'asusu | 50-60 | 30 sec | |
| Fa'aopoopoga | 72 | 30-60 sec/kb | |
| Fa'aopoopoga mulimuli | 72 | 10 minu | 1 |
Fa'aaliga:
1) Fa'aoga fa'atusa: 50-200ng genomic DNA;0.1-10ng plasmid DNA.
2) Mg2+fa'atonuga: O lenei oloa e aofia ai le 3mM o MgCl2, talafeagai mo le tele o tali PCR.
3) vevela vevela: Fa'amolemole va'ai i le tau Tm fa'ata'ita'i 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, 60 sec/kb ua fautuaina.
Fa'amatalaga
1.PCR oloa ma 2× PCR Master Mix e le talafeagai mo polyacrylamide gel electrophoresis.
2.Mo lou saogalemu ma lou soifua maloloina, fa'amolemole fai ni ofu talaloa ma totigilima fa'aaoga mo le taotoga.
3.E fa'aoga mo su'esu'ega NA'O!
