Wild Taq DNA Polymerase
Taq DNA Polymerase ndi DNA polymerase yotentha kuchokera ku Thermus aquaticus YT-1, yomwe ili ndi 5'→ 3' polymerase ntchito ndi 5' flap endonuclease ntchito.
Zigawo
| Chigawo | HC1010A-01 | HC1010A-02 | HC1010A-03 | HC1010A-04 |
| 10 × Taq Buffer | 2 × 1 mL | 2 × 10 mL | 2 × 50 mL | 5 × 200 ml |
| Taq DNA Polymerase (5 U/μL) | 0.1 ml pa | 1 ml | 5ml pa | 5 × 10 mL |
Mkhalidwe Wosungira
Kuyenda pansi pa 0 ° C ndikusungidwa pa -25 ° C ~ -15 ° C.
Tanthauzo la Chigawo
Chigawo chimodzi chimatanthauzidwa ngati kuchuluka kwa enzyme yomwe imaphatikiza 15 nmol ya dNTP muzinthu zosasungunuka za asidi mu mphindi 30 pa 75 ° C.
Kuwongolera Kwabwino
1.Protein Purity Assay (SDS-PAGE):Kuyera kwa Taq DNA polymerase kunali ≥95% kutsimikiziridwa ndi kusanthula kwa SDS-PAGE.
2.EndOnuclease Ntchito:Kuchepa kwa 5 U ya Taq DNA polymerase yokhala ndi 1 μg λDNA kwa maola 16 pa 37 ℃ kumapangitsa kuti pasakhale kuwonongeka komwe kwatsimikiziridwa.
3.Ntchito ya Exonuclease:Kuchepa kwa 5 U ya Taq DNA polymerase yokhala ndi 1 μg λ -Hind Ⅲ digest DNA kwa maola 16 pa 37 ℃ kumapangitsa kuti pasakhale kuwonongeka komwe kwatsimikiziridwa.
4.Ntchito ya Nickase:Kuchepa kwa 5 U ya Taq DNA polymerase yokhala ndi 1 μg pBR322 DNA kwa maola 16 pa 37 ° C kumapangitsa kuti pasakhale kuwonongeka komwe kumatsimikiziridwa.
5.Ntchito ya RNase:Kuchepa kwa 5 U ya Taq DNA polymerase yokhala ndi 1.6 μg MS2 RNA kwa maola 16 pa 37 ° C kumapangitsa kuti pasakhale kuwonongeka komwe kumadziwika.
6.E. koliDNA:5 U of Taq DNA polymerase amawunikidwa kuti ali ndi E. coli genomic DNA pogwiritsa ntchito TaqMan qPCR yokhala ndi zoyambira za E. coli 16S rRNA locus.Kuwonongeka kwa E. coli genomic DNA ndi ≤1 Copy.
7.Kukulitsa kwa PCR (5.0 kb Lambda DNA)- Mawonekedwe a 50 µL okhala ndi 5 ng Lambda DNA yokhala ndi mayunitsi 5 a Taq DNA Polymerase pamizere 25 ya kukulitsa kwa PCR kumabweretsa zomwe zikuyembekezeredwa 5.0 kb.
Kukonzekera kwa Reaction
| Zigawo | Voliyumu |
| DNA templatea | kusankha |
| 10 μM Yoyambira Patsogolo | 1 ml |
| 10 μM Reverse Primer | 1 ml |
| dNTP Mix (10mM iliyonse) | 1 ml |
| 10 × Taq Buffer | 5 ml |
| Taq DNA Polymeraseb | 0.25 μL |
| Madzi opanda nyukiliya | Mpaka 50 μL |
Ndemanga:
1) The mulingo woyenera kwambiri anachita ndende zosiyanasiyana zidindo ndi osiyana.Gome lotsatirali likuwonetsa kugwiritsa ntchito template yovomerezeka ya 50 μL reaction system.
| DNA | Ndalama |
| Genomic | 1 ng-1 μg |
| Plasmid kapena Viral | 1 p.1 ndi |
2) Kuchulukana koyenera kwa Taq DNA Polymerase kumatha kuchoka pa 0.25 µL ~ 1 µL m'mapulogalamu apadera.
Zomwe anachitaPulogalamu
| Khwerero | Kutentha(°C) | Nthawi | Zozungulira |
| Denaturation koyambaa | 95 ℃ | 5 mins | - |
| Denaturation | 95 ℃ | 15-30 s | 30-35 kuzungulira |
| Annealingb | 60 ℃ | 15 s | |
| Kuwonjezera | 72 ℃ | 1kb/mphindi | |
| Final Extension | 72 ℃ | 5 mins | - |
Ndemanga:
1) Chikhalidwe choyambirira cha denaturation ndi choyenera kuchitapo kanthu kokulirapo ndipo chimatha kusinthidwa molingana ndi zovuta zamapangidwe a template.Ngati mawonekedwe a template ndi ovuta, nthawi ya pre-denaturation imatha kukulitsidwa mpaka 5 - 10mins kuti musinthe mawonekedwe oyambira.
2) Kutentha kwa annealing kuyenera kusinthidwa molingana ndi mtengo wa Tm woyambira, womwe nthawi zambiri umayikidwa ku 3 ~ 5 ℃ kutsika kuposa mtengo wa Tm woyambira;Kwa ma template ovuta, ndikofunikira kusintha kutentha kwa annealing ndikuwonjezera nthawi kuti mukwaniritse bwino kukulitsa.
