Hotstart Taq ADN Polymerase
Hot Start Taq ADN Polymerase (Guhindura Antibody) ni hot-itangira gushyuha cyane ya polymerase ya ADN ivuye muri Thermus aquaticus YT-1 ， ifite ibikorwa bya 5 ′ → 3 ′ polymerase nibikorwa bya 5´ flap endonuclease.Gushyushya-gutangira Taq ADN polymerase ni polymerase ya Taq ADN yahinduwe na antibodiyite za termolabile Taq.Guhindura Antibody byongereye umwihariko, ibyiyumvo, numusaruro wa PCR.
Ibigize
| Ibigize | HC1012A-01 | HC1012A-02 | HC1012A-03 | HC1012A-04 |
| 5 × HC Taq Buffer | 4 × 1 mL | 4 × 10 mL | 4 × 50 mL | 5 × 400 mL |
| Tangira Ashyushye Taq ADN Polymerase (Antibody yahinduwe) (5 U / μL) | 0.1 mL | 1 mL | 5 mL | 10 × 5 mL |
Porogaramu
10 mM Tris-HCl (pH 7.4 kuri 25 ℃), 100 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, 0.5% Hagati ya 20, 0.5% IGEPALCA-630 na 50% Glycerol.
Imiterere y'Ububiko
Ubwikorezi buri munsi ya 0 ° C kandi bubikwa kuri -25 ° C ~ -15 ° C.
Igisobanuro
Igice kimwe gisobanurwa nkubunini bwa enzyme yinjiza nmol 15 ya dNTP mubintu bitangirika aside muminota 30 kuri 75 ° C.
Kugenzura ubuziranenge
1.Endonuclease Igikorwa:Inkubasi ya 20 U ya enzyme hamwe na 4 μg pUC19 ADN yamasaha 4 kuri 37 ℃ byatumye nta kwangirika kugaragara kwa ADN nkuko byagenwe na gel electrophorei.
2.5 kb Lambda PCR:25 Cycle ya PCR yongerewe imbaraga 5 ng ya ADN ya Lambda hamwe na 1.25 bya Taq DNA Polymerase imbere ya 200 µM dNTPs na 0.2 µM primers bivamo ibicuruzwa biteganijwe 5 kb.
3.Exonuclease Igikorwa:Inkubasi ya 50 µl reaction irimo byibura 12.5 U ya Taq DNA Polymerase hamwe na 10 nmol 5´-FAM oligonucleotide muminota 30 kuri 37 ℃ ntabwo itanga kwangirika kugaragara.
4.Igikorwa cya RNase:Inkubasi ya 10 µL reaction irimo 20 U ya enzyme hamwe na 1μg ya transcript ya RNA mumasaha 2 kuri 37 ° C byatumye nta kwangirika kugaragara kwa RNA nkuko byagenwe na gel electrophorei.
5.Gutangiza ubushyuhe:Oya.
Sisitemu yo Kwitabira
| Ibigize | Umubumbe |
| Icyitegererezo ADNa | bidashoboka |
| 10 μM Imbere Imbere | 0.5 μL |
| 10 μM Guhindura Primer | 0.5 μL |
| DNTP ivanga (10mM buri umwe) | 0.5 μL |
| 5 × HC Taq Buffer | 5 μL |
| Taq ADN Polymeraseb(5U / μL) | 0.125 μL |
| Amazi adafite ingufu | Kugera kuri 25 μL |
Inyandiko:
1) a.
| ADN | Umubare |
| Genomic | 1 ng-1 μg |
| Plasmid cyangwa Virusi | 1 pg-1 ng |
2) b.Ibyiza bya Taq ADN Polymerase irashobora kuva kuri 5-50 / mL (0.1-0.5 ibice / 25 µL reaction) mubikorwa byihariye.
Umukino wo gusiganwa ku magare
PCR
| Intambwe | Ubushyuhe(° C.) | Igihe | Amagare |
| Gutandukana kwamberea | 95 ℃ | 1-3min | - |
| Gutandukana | 95 ℃ | 15-30 s | 30-35 Amagare |
| Annealingb | 45-68 ℃ | 15-60 s | |
| Kwagura | 68 ℃ | 1kb / min | |
| Kwagura kwanyuma | 68 ℃ | 5min | - |
Inyandiko:
1) Gutandukana kwambere kwiminota 1 kuri 95 ° C birahagije kuri amplification nyinshi.Kubishusho bigoye, birasabwa gutandukana birebire bya 2-3min kuri 95 ° C.Hamwe na koloni PCR, birashoboka ko 5mins ibanza gutandukana kuri 95 ° C.
2) Intambwe ya annealing mubisanzwe ni 15-60 s.Ubushyuhe bwa Annealing bushingiye kuri Tm ya primer couple kandi mubisanzwe ni 45-68 ℃.
