I-Hotstart Taq DNA Polymerase
I-Hot Start Taq DNA Polymerase (Ukuguqulwa kwe-Antibody) sisiqalo esishushu se-thermostable DNA polymerase evela kwi-Thermus aquaticus YT-1, enomsebenzi we-5′→3′ wepolymerase kunye ne-5′ flap endonuclease.I-polymerase ye-Taq DNA eshushu iqala i-Taq DNA yepolymerase eguqulwe nge-thermolabile Taq antibodies.Ukuguqulwa kwe-Antibody kwandisa ukucaca, uvakalelo, kunye nesivuno se-PCR.
Amacandelo
| Icandelo | HC1012A-01 | HC1012A-02 | HC1012A-03 | HC1012A-04 |
| 5×HC Taq Buffer | 4×1 mL | 4×10 mL | 4×50 mL | 5×400 mL |
| Isiqalo esishushu iTaq DNA Polymerase (i-Antibody ilungisiwe) (5 U/μL) | 0.1 ml | 1 ml | 5 ml | 10×5 mL |
Usetyenziso
10 mM Tris-HCl (pH 7.4 ku-25℃), 100 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, 0.5% Tween20, 0.5% IGEPALCA-630 kunye ne-50% Glycerol.
Imeko yoGcino
Ukuthutha ngaphantsi kwe-0°C kwaye igcinwe kwi-25°C~-15°C.
Inkcazelo yeyunithi
Iyunithi enye ichazwa njengobungakanani be-enzyme edibanisa i-15 nmol ye-dNTP kwizinto ezingenakunyibilika ze-asidi kwimizuzu engama-30 kwi-75 ° C.
Ulawulo lwemeko
1.EndOnuclease Umsebenzi:Ukufukanyelwa kwe-20 U ye-enzyme ene-4 μg pUC19 DNA kwiiyure ezi-4 kwi-37℃ kubangele kungabikho ukuthotywa okubonakalayo kwe-DNA njengoko kumiselwe yi-gel electrophoresis.
2.5 kb Lambda PCR:Imijikelezo engama-25 ye-PCR yokukhulisa i-5 ng Lambda DNA eneyunithi ye-1.25 ye-Taq DNA Polymerase kubukho be-200 µM dNTPs kunye ne-0.2 µM yokuqala iphumela kwimveliso elindelekileyo ye-5 kb.
3.Umsebenzi we-Exonuclease:Ukufukanyelwa kwe-50 µl reaction equlathe ubuncinci be-12.5 U ye-Taq DNA Polymerase ene-10 nmol 5´-FAM oligonucleotide kangangemizuzu engama-30 ngama-37℃ akuvelisi ukuthotywa kubonwa.
4.Umsebenzi we-RNase:Ukufukanyelwa kwe-10 µL reaction equlathe i-20 U ye-enzyme ene-1μg ye-RNA ekhutshelweyo kwiiyure ezi-2 kuma-37°C kubangele kungabikho kuthotywa kubonwayo kwe-RNA njengoko kumiselwe yijeli ye-electrophoresis.
5.Ukungasebenzisi Ubushushu:Hayi.
Indlela yokusabela
| Amacandelo | Umthamo |
| Isakhelo seDNAa | ngokuzikhethela |
| 10 μM Phambili iPrimer | 0.5 μL |
| 10 μM Ukubuyisela umva iPrimer | 0.5 μL |
| I-dNTP Mix (10mM nganye) | 0.5 μL |
| 5×HC Taq Buffer | 5 μL |
| Taq DNA Polymeraseb(5U/μL) | 0.125 μL |
| Amanzi angenanyukliya | Ukuya kuthi ga kwi-25 μL |
Amanqaku:
1) a.
| DNA | Isixa |
| Genomic | 1 ng-1 μg |
| I-Plasmid okanye iViral | 1 iphe-1 ng |
2) b.Olona xinzelelo luphezulu lwe-Taq DNA Polymerase lunokusuka kwi-5-50 units/mL (0.1-0.5 units/25 µL reaction) kwizicelo ezikhethekileyo.
Iprothokholi yebhayisekile eshushu
I-PCR
| Inyathelo | Ubushushu(°C) | Ixesha | Imijikelo |
| I-denaturation yokuqalaa | 95 ℃ | 1-3mins | - |
| I-Denaturation | 95 ℃ | 15-30 s | 30-35 Imijikelezo |
| Ukuhlaziyab | 45-68 ℃ | 15-60 s | |
| Ulwandiso | 68 ℃ | 1kb/min | |
| Ukwandiswa kokugqibela | 68 ℃ | 5 imiz | - |
Amanqaku:
I-1) I-denaturation yokuqala ye-1 min kwi-95 ° C yanele ubuninzi be-amplification.Kwiitemplates ezinzima, i-denaturation ende ye-2-3mins kwi-95 ° C iyacetyiswa.Ngekholoni ye-PCR, i-5mins yokuqala ye-denaturation kwi-95 ° C iyacetyiswa.
2) Inyathelo le-annealing ngokuqhelekileyo yi-15-60 s.Ubushushu be-Anealing busekwe kwi-Tm yeprimer pair kwaye ngokuqhelekileyo yi-45-68℃.
