Bst 2.0 DNA Polymerase (Glycerol yaulere)
Bst DNA polymerase V2 imachokera ku Bacillus stearothermophilus DNA Polymerase I, yomwe ili ndi 5'→ 3' DNA polymerase ntchito ndi ntchito yamphamvu yowonjezera unyolo, koma palibe 5′→3′ exonuclease ntchito.Bst DNA Polymerase V2 ndiyoyenera kusamuka kwa strand, isothermal amplification LAMP (Loop mediated isothermal amplification) ndikutsatizana mwachangu.
Zigawo
| Chigawo | Chithunzi cha HC5005A-01 | Chithunzi cha HC5005A-02 | Chithunzi cha HC5005A-03 |
| BstDNApolymerase V2(Glycerol-free) (8U/μL) | 0.2 ml pa | 1 ml | 10 ml pa |
| 10 × HC Bst V2 Buffer | 1.5 ml | 2 × 1.5 mL | 3 × 10 mL |
| MgSO4(100mM) | 1.5 ml | 2 × 1.5 mL | 2 × 10 mL |
Mapulogalamu
1.LAMP isothermal amplification
2.DNA strand single displacement reaction
3.High GC gene sequencing
4.Kutsata kwa DNA kwa mlingo wa nanogram.
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 25 nmol ya dNTP muzinthu zosasungunuka za asidi mu mphindi 30 pa 65 ° C.
Kuwongolera Kwabwino
1.Protein Purity Assay (SDS-PAGE):Kuyera kwa Bst DNA polymerase V2 ndi ≥99% yotsimikiziridwa ndi kusanthula kwa SDS-PAGE pogwiritsa ntchito kuzindikira kwa Coomassie Blue.
2.Ntchito ya Exonuclease:Kuyika kwa 50 μL reaction yokhala ndi osachepera 8 U ya Bst DNA polymerase V2 yokhala ndi 1 μg λ -Hind Ⅲ digest DNA kwa maola 16 pa 37 ℃ kumapangitsa kuti pasakhale kuwonongeka komwe kwatsimikiziridwa.
3.Ntchito ya Nickase:Kuyika kwa 50 μL reaction yokhala ndi osachepera 8 U ya Bst DNA polymerase V2 yokhala ndi 1 μg pBR322 DNA kwa maola 16 pa 37°C kumapangitsa kuti pasakhale kuwonongeka kodziwika monga momwe zatsimikizidwira.
4.Ntchito ya RNase:Kuyika kwa 50 μL reaction yokhala ndi osachepera 8 U ya Bst DNA polymerase V2 yokhala ndi 1.6 μg MS2 RNA kwa maola 16 pa 37°C kumapangitsa kuti pasakhale kuwonongeka kodziwika monga momwe zatsimikizidwira.
5.E. coli DNA:120 U of Bst DNA polymerase V2 imawunikiridwa ngati pali 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.
Kusintha kwa LAMP
| Zigawo | 25μL |
| 10 × HC Bst V2 Buffer | 2.5 μL |
| MgSO4 (100mM) | 1.5 μL |
| dNTPs (10mM iliyonse) | 3.5 μl |
| SYTO™ 16 Green (25×)a | 1.0 μL |
| Kusakaniza koyambirirab | 6 ml |
| Bst DNA Polymerase V2 (Glycerol-free) (8 U/uL) | 1 ml |
| Template | × μl ndi |
| ddH₂O | Mpaka 25 μL |
Ndemanga:
1) a.SYTOTM 16 Green (25 ×): Malingana ndi zosowa zoyesera, utoto wina ukhoza kugwiritsidwa ntchito m'malo;
2) b.Kusakaniza koyamba: kupezedwa posakaniza 20 µ M FIP, 20 µ M BIP, 2.5 µ M F3, 2.5 µ M B3, 5 µ M LF, 5 µ M LB ndi mabuku ena.
Zochita ndi Mkhalidwe
1 × HC Bst V2 Buffer, kutentha kwa ma incubation kuli pakati pa 60°C ndi 65°C.
Kutentha Kutentha
80 ° C, mphindi 20
