Hotstart Taq DNA Polymerase
Hot Start Taq DNA Polymerase (Antibody modification) ke DNA polymerase e chesang e qalang thermostable e tsoang ho Thermus aquaticus YT-1, e nang le ts'ebetso ea 5′→3′ polymerase le ts'ebetso ea 5' flap endonuclease.Taq DNA polymerase e qalang e chesang ke Taq DNA polymerase e fetotsoeng ke li-antibodies tsa thermolabile Taq.Phetoho ea li-antibody e ekelitse boits'oaro, kutloisiso, le tlhahiso ea PCR.
Likaroloana
| Karolo | HC1012A-01 | HC1012A-02 | HC1012A-03 | HC1012A-04 |
| 5×HC Taq Buffer | 4×1 mL | 4×10 mL | 4×50 mL | 5 × 400 mL |
| Hot Start Taq DNA Polymerase (Antibody e fetotsoe) (5 U/μL) | 0.1 mL | 1 mL | 5 mL | 10 × 5 mL |
Lisebelisoa
10 mM Tris-HCl (pH 7.4 ho 25℃), 100 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, 0.5% Tween20, 0.5% IGEPALCA-630 le 50% Glycerol.
Boemo ba polokelo
Lipalangoang ka tlas'a 0°C 'me li bolokoe ho -25°C~-15°C.
Tlhaloso ea Yuniti
Yuniti e le 'ngoe e hlalosoa e le palo ea enzyme e kenyelletsang 15 nmol ea dNTP linthong tse sa keneng tsa asiti ka metsotso e 30 ho 75°C.
Taolo ea Boleng
1.EndOnuclease Mosebetsi:Ho kenngoa ha 20 U ea enzyme e nang le 4 μg pUC19 DNA bakeng sa lihora tse 4 ho 37 ℃ ho entse hore ho se be le ho senyeha ho bonahalang ha DNA joalokaha ho laetsoe ke gel electrophoresis.
2.5kb Lambda PCR:25 Cycles of PCR amplification of 5 ng Lambda DNA with 1.25 units of Taq DNA Polymerase ka boteng ba 200 µM dNTPs le 0.2 µM primers e fella ka sehlahisoa se lebelletsoeng sa 5 kb.
3.Ts'ebetso ea Exonuclease:Ho kenyeletswa ha 50 µl reaction e nang le bonyane ba 12.5 U ya Taq DNA Polymerase e nang le 10 nmol 5'-FAM oligonucleotide bakeng sa metsotso e 30 ho 37℃ ha e fane ka tshenyeho e bonahalang.
4.Mosebetsi oa RNase:Ho kenyeletsoa ha 10 µL reaction e nang le 20 U ea enzyme e nang le 1μg ea likopi tsa RNA bakeng sa lihora tse 2 ho 37 ° C ho entse hore ho se be le ho senyeha ho bonahalang ha RNA joalokaha ho laetsoe ke gel electrophoresis.
5.Ho bulela Mocheso:Che.
Reaction System
| Likaroloana | Bolumo |
| DNA ea Setšoantšoa | boikhethelo |
| 10 μM Pele Pele | 0.5 μL |
| 10 μM Reverse Primer | 0.5 μL |
| dNTP Mix (10mM e 'ngoe le e 'ngoe) | 0.5 μL |
| 5×HC Taq Buffer | 5 μL |
| Taq DNA Polymeraseb(5U/μL) | 0.125 μL |
| Metsi a se nang nyutlelie | Ho fihlela ho 25 μL |
Lintlha:
1) a.
| DNA | Chelete |
| Genomic | 1 ng-1 μg |
| Plasmid kapa kokoana-hloko | 1 leq-1 ng |
2) b.Khokahano e nepahetseng ea Taq DNA Polymerase e ka tloha ho 5-50 units/mL (0.1-0.5 units/25 μL reaction) lits'ebetsong tse ikhethileng.
Thermal cycling protocol
PCR
| Mohato | Mocheso(°C) | Nako | Lipotoloho |
| Denaturation ea pelea | 95 ℃ | 1-3 mets | - |
| Denaturation | 95 ℃ | 15-30 s | 30-35 Lipotoloho |
| Annealingb | 45-68 ℃ | 15-60 s | |
| Katoloso | 68 ℃ | 1kb/mots | |
| Keketso ea ho Qetela | 68 ℃ | 5 mets | - |
Lintlha:
1) Denaturation ea pele ea 1 min ho 95 ° C e lekane bakeng sa li-amplification tse ngata.Bakeng sa litempele tse thata, ho buelloa nako e telele ea 2-3mins ho 95 ° C.Ka kolone ea PCR, ho khothaletsoa ho hlahisa maikutlo a pele a 5mins ho 95 ° C.
2) Mohato oa annealing hangata ke 15-60 s.Thempereichara ea Annealing e thehiloe ho Tm ea paramente ea pele mme hangata ke 45-68 ℃.
