Hotstart Taq DNA Polymerase
Kub Start Taq DNA Polymerase (Antibody modification) yog kub-pib thermostable DNA polymerase los ntawm Thermus aquaticus YT-1, uas muaj 5′→ 3′ polymerase kev ua thiab 5′ flap endonuclease kev ua.Qhov kub pib Taq DNA polymerase yog Taq DNA polymerase uas hloov kho los ntawm cov tshuaj tiv thaiv thermolabile Taq.Kev hloov kho cov tshuaj tiv thaiv tau nce qhov tshwj xeeb, rhiab heev, thiab tawm los ntawm PCR.
Cheebtsam
| Cheebtsam | HC10 12 A-01 | HC10 12 A-02 | HC10 12 A-03 | HC10 12 A-04 |
| 5 × HC Taq Buffer | 4 x 1ml | 4 × 10 ml | 4 × 50 ml | 5 × 400 ml |
| Kub Pib Taq DNA Polymerase (Antibody hloov kho) (5 U / μL) | 0.1ml ib | 1 ml ib | 5 ml ib | 10 × 5 mL |
Daim ntawv thov
10 mM Tris-HCl (pH 7.4 ntawm 25 ℃), 100 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, 0.5% Tween20, 0.5% IGEPALCA-630 thiab 50% Glycerol.
Kev cia khoom
Kev thauj mus los hauv qab 0 ° C thiab khaws cia ntawm -25 ° C ~ -15 ° C.
Unit txhais
Ib chav tsev yog txhais raws li tus nqi ntawm cov enzyme uas suav nrog 15 nmol ntawm dNTP rau hauv cov khoom siv acid insoluble hauv 30 feeb ntawm 75 ° C.
Kev Tswj Xyuas Zoo
1.Endonuclease kev ua:Incubation ntawm 20 U ntawm enzyme nrog 4 μg pUC19 DNA rau 4 teev ntawm 37 ℃ ua rau tsis pom qhov degradation ntawm DNA raws li kev txiav txim los ntawm gel electrophoresis.
2.5 kb Lambda PCR:25 Lub voj voog ntawm PCR amplification ntawm 5 ng Lambda DNA nrog 1.25 units ntawm Taq DNA Polymerase nyob rau hauv lub xub ntiag ntawm 200 µM dNTPs thiab 0.2 µM primers tshwm sim nyob rau hauv cov khoom xav tau 5 kb.
3.Kev Ua Haujlwm Exonuclease:Incubation ntawm 50 µl cov tshuaj tiv thaiv uas muaj qhov tsawg kawg nkaus ntawm 12.5 U ntawm Taq DNA Polymerase nrog 10 nmol 5'-FAM oligonucleotide rau 30 feeb ntawm 37 ℃ yields tsis pom degradation.
4.RNase Kev Ua Haujlwm:Incubation ntawm 10 µL cov tshuaj tiv thaiv uas muaj 20 U ntawm enzyme nrog 1μg ntawm RNA cov ntaub ntawv pov thawj rau 2 teev ntawm 37 ° C ua rau tsis pom qhov degradation ntawm RNA raws li kev txiav txim los ntawm gel electrophoresis.
5.Thaum tshav kub kub inactivation:Tsis muaj.
Reaction System
| Cheebtsam | Ntim |
| Template DNAa | xaiv tau |
| 10 μM Forward Primer | 0,5l ua |
| 10 μM Rov qab Primer | 0,5l ua |
| dNTP Mix (10mM txhua) | 0,5l ua |
| 5 × HC Taq Buffer | 5l miv |
| Cov DNA Polymeraseb(5U / μL) | 0.125 mL |
| Cov dej tsis muaj Nuclease | Txog li 25 μL |
Nco tseg:
1) a.
| DNA | Tus nqi |
| Genomic | 1 ng - 1 mg |
| Plasmid los yog Viral | 1 pg-1 ib |
2) ib.Qhov zoo tshaj plaws concentration ntawm Taq DNA Polymerase tej zaum yuav txawv ntawm 5-50 units / mL (0.1-0.5 units / 25 µL cov tshuaj tiv thaiv) hauv cov ntawv tshwj xeeb.
Thermal cycling raws tu qauv
PCR
| Kauj ruam | Kub(°C) | Sijhawm | Lub voj voog |
| Thawj denaturationa | 95 ℃ | 1-3 feeb | - |
| Denaturation | 95 ℃ | 15-30 s., kuv | 30-35 Cycle |
| Annealingb | 45-68 ℃ | 15-60 s., kuv | |
| Txuas ntxiv | 68 ℃ | 1 kb/min | |
| Kawg Extension | 68 ℃ | 5 feeb | - |
Nco tseg:
1) Qhov pib denaturation ntawm 1 min ntawm 95 ° C yog txaus rau feem ntau amplifications.Rau cov qauv nyuaj, qhov ntev denaturation ntawm 2-3mins ntawm 95 ° C yog pom zoo.Nrog colony PCR, thawj 5mins denaturation ntawm 95 ° C raug pom zoo.
2) Cov kauj ruam annealing feem ntau yog 15-60 s.Annealing kub yog raws li Tm ntawm primer khub thiab feem ntau yog 45-68 ℃.
