Hotstart Taq DNA Polymerase
Hot Start Taq DNA Polymerase (Kusintha kwa Antibody) ndi DNA polymerase yotentha yoyambira ku Thermus aquaticus YT-1, yomwe imakhala ndi 5'→ 3′ polymerase zochita komanso 5' flap endonuclease.Taq DNA polymerase yotentha yoyambira ndi Taq DNA polymerase yomwe imasinthidwa ndi ma antibodies a thermolabile Taq.Kusintha kwa ma antibodies kumawonjezera kutsimikizika, kukhudzika, ndi zokolola za PCR.
Zigawo
| Chigawo | HC1012A-01 | HC1012A-02 | HC1012A-03 | HC1012A-04 |
| 5 × HC Taq Buffer | 4 × 1 mL | 4 × 10 mL | 4 × 50 ml | 5 × 400 mL |
| Hot Start Taq DNA Polymerase (Antibody Yosinthidwa) (5 U/μL) | 0.1 ml pa | 1 ml | 5ml pa | 10 × 5 mL |
Mapulogalamu
10 mM Tris-HCl (pH 7.4 pa 25℃), 100 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, 0.5% Tween20, 0.5% IGEPALCA-630 ndi 50% Glycerol.
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.EndOnuclease Ntchito:Kuphatikizika kwa 20 U ya enzyme yokhala ndi 4 μg pUC19 DNA kwa maola 4 pa 37 ℃ sikunapangitse kuwonongeka kwa DNA komwe kumatsimikiziridwa ndi gel electrophoresis.
2.5 KB Lambda PCR:25 Cycles of PCR amplification of 5 ng Lambda DNA with 1.25 units of Taq DNA Polymerase pamaso pa 200 µM dNTPs ndi 0.2 µM primers zimabweretsa 5 kb yoyembekezeredwa.
3.Ntchito ya Exonuclease:Kuyika kwa 50 µl reaction yokhala ndi osachepera 12.5 U ya Taq DNA Polymerase yokhala ndi 10 nmol 5'-FAM oligonucleotide kwa mphindi 30 pa 37 ℃ sikutulutsa kuwonongeka kowonekera.
4.Ntchito ya RNase:Kuphatikizika kwa 10 µL reaction yokhala ndi 20 U ya enzyme yokhala ndi 1μg ya zolembedwa za RNA kwa maola 2 pa 37 ° C sikunapangitse kuwonongeka kwa RNA komwe kumatsimikiziridwa ndi gel electrophoresis.
5.Kusiya Kutentha:Ayi.
Reaction System
| Zigawo | Voliyumu |
| DNA templatea | kusankha |
| 10 μM Yoyambira Patsogolo | 0.5 μL |
| 10 μM Reverse Primer | 0.5 μL |
| dNTP Mix (10mM iliyonse) | 0.5 μL |
| 5 × HC Taq Buffer | 5 ml |
| Taq DNA Polymeraseb(5U/μL) | 0.125 μL |
| Madzi opanda nyukiliya | Mpaka 25 μL |
Ndemanga:
1) a.
| DNA | Ndalama |
| Genomic | 1 ng-1 μg |
| Plasmid kapena Viral | 1 p.1 ndi |
2) b.Mulingo woyenera kwambiri wa Taq DNA Polymerase ukhoza kuyambira 5-50 mayunitsi/mL (0.1-0.5 mayunitsi/25 µL reaction) m'mapulogalamu apadera.
Thermal cycling protocol
PCR
| Khwerero | Kutentha(°C) | Nthawi | Zozungulira |
| Denaturation koyambaa | 95 ℃ | 1-3 mphindi | - |
| Denaturation | 95 ℃ | 15-30 s | 30-35 kuzungulira |
| Annealingb | 45-68 ℃ | 15-60 s | |
| Kuwonjezera | 68 ℃ | 1kb/mphindi | |
| Final Extension | 68 ℃ | 5 mins | - |
Ndemanga:
1) Kuwonetsa koyamba kwa 1 min pa 95 ° C ndikokwanira kukulitsa zambiri.Kwa ma templates ovuta, kutanthauzira kwakutali kwa 2-3mins pa 95 ° C ndikulimbikitsidwa.Ndi PCR yamtundu, kutanthauzira koyambirira kwa 5mins pa 95 ° C kumalimbikitsidwa.
2) Gawo la annealing nthawi zambiri ndi 15-60 s.Kutentha kwa Annealing kumachokera ku Tm ya primer pair ndipo nthawi zambiri ndi 45-68 ℃.
