Ishamba rya Taq DNA Polymerase
Taq ADN Polymerase ni polymerase ya ADN ikomoka kuri Thermus aquaticus YT-1, ifite ibikorwa bya 5 ′ → 3 ′ polymerase nigikorwa cya 5´ flap endonuclease.
Ibigize
| Ibigize | HC1010A-01 | HC1010A-02 | HC1010A-03 | HC1010A-04 |
| 10 Taf Buffer | 2 × 1 mL | 2 × 10 mL | 2 × 50 mL | 5 × 200 mL |
| Taq ADN Polymerase (5 U / μL) | 0.1 mL | 1 mL | 5 mL | 5 × 10 mL |
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.Isuzuma rya poroteyine (SDS-PAGE):Ubuziranenge bwa Taq ADN polymerase yagenwe ≥ 95% byagenwe nisesengura rya SDS-PAGE.
2.Endonuclease Igikorwa:Nibura 5 U ya Taq ADN polymerase hamwe na 1 μg λDNA kumasaha 16 kuri 37 ℃ bivamo nta kwangirika kugaragara nkuko byagenwe.
3.Exonuclease Igikorwa:Nibura 5 U ya Taq ADN polymerase hamwe na 1 μg λ -Hind Ⅲ igogora ADN amasaha 16 kuri 37 ℃ bivamo nta kwangirika kugaragara nkuko byagenwe.
4.Igikorwa cya Nickase:Nibura 5 U ya Taq ADN polymerase hamwe na 1 μg pBR322 ADN mumasaha 16 kuri 37 ° C bivamo nta kwangirika kugaragara nkuko byagenwe.
5.Igikorwa cya RNase:Nibura 5 U ya Taq ADN polymerase hamwe na 1,6 μg MS2 RNA mumasaha 16 kuri 37 ° C bivamo nta kwangirika kugaragara nkuko byagenwe.
6.E. coliADN:5 U ya Taq ADN polymerase isuzumwa kugirango ADN genomic E. coli ikoreshe TaqMan qPCR hamwe na primers yihariye ya E. coli 16S rRNA.E. coli genomic ADN yanduye ni ≤1 Gukoporora.
7.Kwiyongera kwa PCR (5.0 kb ADN ya Lambda)- 50 µL reaction irimo 5 ng ya ADN ya Lambda hamwe na 5 ya Taq ADN Polymerase kuri cycle 25 ya PCR yongerewe ibisubizo mubicuruzwa biteganijwe 5.0 kb.
Gushiraho
| Ibigize | Umubumbe |
| Icyitegererezo ADNa | bidashoboka |
| 10 μM Imbere Imbere | 1 μL |
| 10 μM Guhindura Primer | 1 μL |
| DNTP ivanga (10mM buri umwe) | 1 μL |
| 10 Taf Buffer | 5 μL |
| Taq ADN Polymeraseb | 0,25 μL |
| Amazi adafite ingufu | Kugera kuri 50 μL |
Inyandiko:
1) Ibyiza bya reaction yibitekerezo bitandukanye biratandukanye.Imbonerahamwe ikurikira irerekana icyitegererezo cyakoreshejwe cya 50 µL reaction ya sisitemu.
| ADN | Umubare |
| Genomic | 1 ng-1 μg |
| Plasmid cyangwa Virusi | 1 pg-1 ng |
2) Ibyiza bya Taq DNA Polymerase irashobora kuva kuri 0.25 µL ~ 1 µL mubikorwa byihariye.
IgisubizoGahunda
| Intambwe | Ubushyuhe(° C.) | Igihe | Amagare |
| Gutandukana kwamberea | 95 ℃ | 5min | - |
| Gutandukana | 95 ℃ | 15-30 s | 30-35 Amagare |
| Annealingb | 60 ℃ | 15 s | |
| Kwagura | 72 ℃ | 1kb / min | |
| Kwagura kwanyuma | 72 ℃ | 5min | - |
Inyandiko:
1) Imiterere yambere yo gutandukana irakwiriye kubantu benshi bongera imbaraga kandi irashobora guhindurwa ukurikije imiterere yimiterere.Niba inyandikorugero yuburyo bugoye, igihe cyabanjirije gutandukana gishobora kwongerwa kugeza 5 - 10min kugirango tunoze ingaruka zambere.
2) Ubushyuhe bwa annealing bugomba guhinduka ukurikije agaciro ka Tm ya primer, ubusanzwe yashyizwe kuri 3 ~ 5 ℃ munsi ya Tm agaciro ka primer;Kuri templates zigoye, birakenewe guhindura ubushyuhe bwa annealing no kongera igihe cyo kwaguka kugirango ugere kuri amplification neza.
