I-Hotstart Taq DNA Polymerase
I-Hot Start Taq DNA Polymerase (Ukuguqulwa kwe-Antibody) iyi-polymerase ye-DNA eqala ukushisa eshisayo evela ku-Thermus aquaticus YT-1, enomsebenzi we-polymerase ongu-5′→3′ kanye nomsebenzi we-endonuclease we-flap ongu-5.I-Taq DNA polymerase eqala ukushisa i-Taq DNA polymerase eshintshwe amasosha omzimba e-thermolabile Taq.Ukuguqulwa kwamasosha omzimba kukhuphule ukucaciswa, ukuzwela, kanye nesivuno se-PCR.
Izingxenye
| Isakhi | HC1012A-01 | HC1012A-02 | HC1012A-03 | HC1012A-04 |
| 5×HC Taq Buffer | 4×1 mL | 4×10 mL | 4×50 mL | 5×400 mL |
| I-Hot Start Taq DNA Polymerase (Amasosha omzimba ashintshiwe) (5 U/μL) | 0.1 mL | 1 mL | 5 ml | 10×5 mL |
Izinhlelo zokusebenza
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 kanye no-50% Glycerol.
Isimo Sesitoreji
Ukuthutha ngaphansi kuka-0°C futhi kugcinwe ku -25°C~-15°C.
Incazelo Yeyunithi
Iyunithi eyodwa ichazwa njengenani le-enzyme elihlanganisa i-15 nmol ye-dNTP kwinto engancibiliki ene-asidi emizuzwini engama-30 ku-75°C.
Ikhwalithi yokulawula
1.EndUmsebenzi we-onuclease:Ukufakwa kwe-20 U ye-enzyme ene-4 μg pUC19 DNA amahora angu-4 ku-37℃ kubangele kungabikho ukuwohloka okubonakalayo kwe-DNA njengoba kunqunywe i-gel electrophoresis.
2.5 KB Lambda PCR:Imijikelezo engu-25 ye-PCR yokukhulisa i-5 ng Lambda DNA enamayunithi angu-1.25 e-Taq DNA Polymerase ebukhoneni buka-200 µM dNTPs kanye nama-primer angu-0.2 µM kuphumela kumkhiqizo olindelwe ongu-5 kb.
3.Umsebenzi we-Exonuclease:Ukufakwa kokusabela okungu-50 µl okuqukethe ubuncane be-12.5 U ye-Taq DNA Polymerase ne-10 nmol 5'-FAM oligonucleotide imizuzu engu-30 ngo-37℃ akukhiqizi ukuwohloka okubonakalayo.
4.Umsebenzi we-RNase:Ukufakwa kokusabela okungu-10 µL okuqukethe u-20 U we-enzyme ene-1μg yemibhalo ye-RNA amahora angu-2 ku-37°C kubangele kungabikho ukuwohloka okubonakalayo kwe-RNA njengoba kunqunywa ijeli electrophoresis.
5.Ukushisa Ukungasebenzi:Cha.
Isistimu yokusabela
| Izingxenye | Ivolumu |
| I-DNA yesifanekisoa | ngokuzikhethela |
| 10 μM Phambili I-Primer | 0.5 μL |
| 10 μM I-Primer Reverse | 0.5 μL |
| I-dNTP Mix (10mM ngayinye) | 0.5 μL |
| 5×HC Taq Buffer | 5 μl |
| I-Taq DNA Polymeraseb(5U/μL) | 0.125 μL |
| Amanzi angenayo i-nuclease | Kufika ku-25 μL |
Amanothi:
1) a.
| I-DNA | Inani |
| I-Genomic | 1 ng-1 μg |
| I-Plasmid noma i-Viral | 1 ikhasi-1 ng |
2) b.Ukuhlushwa okuphelele kwe-Taq DNA Polymerase kungase kusuke ku-5-50 amayunithi/mL (amayunithi angu-0.1-0.5/25 µL ukusabela) ezinhlelweni ezikhethekile.
Iphrothokholi yokuhamba ngebhayisikili eshisayo
I-PCR
| Isinyathelo | Izinga lokushisa(°C) | Isikhathi | Imijikelezo |
| I-denaturation yokuqalaa | 95 ℃ | 1-3 amaminithi | - |
| I-Denaturation | 95 ℃ | 15-30 s | 30-35 Imijikelezo |
| Anealingb | 45-68 ℃ | 15-60 s | |
| Isandiso | 68 ℃ | 1kb/min | |
| Isandiso Sokugcina | 68 ℃ | 5 imiz | - |
Amanothi:
I-1) I-denaturation yokuqala ye-1 min ku-95 ° C yanele ekukhuliseni okuningi.Ngezifanekiso ezinzima, ukuhunyushwa okude kwemizuzu engu-2-3 ku-95°C kuyanconywa.Nge-PCR yekholoni, ukuchazwa kwe-denaturation kokuqala okuyimizuzu emi-5 ku-95°C kuyanconywa.
2) Isinyathelo se-anneal ngokuvamile siyi-15-60 s.Izinga lokushisa le-annealing lisuselwa ku-Tm ye-primer pair futhi ngokuvamile lingama-45-68℃.
