Gutangira neza Bst 2.0 Polymerase ya ADN (Glycerol yubusa)
Bst ADN polymerase V2 yakomotse kuri Bacillus stearothermophilus ADN Polymerase I, ifite 5 ′ → 3 ′ ibikorwa bya polymerase ya ADN nigikorwa gikomeye cyo gusimbuza urunigi, ariko nta gikorwa cya 5 ′ → 3 ′.Bst ADN Polymerase V2 nibyiza cyane muburyo bwo kwimura imirongo, kwimura isothermal amplification LAMP (Loop mediated isothermal amplification) hamwe nuburyo bwihuse.Bst ADN polymerase V2 ni verisiyo ishyushye itangira ishingiye kuri Bst ADN polymerase V2 (HC5005A) yabonetse hakoreshejwe tekinoroji yo guhindura ibintu, ishobora guhagarika ibikorwa bya ADN polymerase mubushyuhe bwicyumba, bityo sisitemu yo kubyitwaramo irashobora gukoreshwa no gutegurwa mubushyuhe bwicyumba kugirango birinde non -Ibisobanuro byihariye kandi bitezimbere imikorere, kandi iyi verisiyo irashobora gukoreshwa.Mubyongeyeho, ibikorwa byayo bisohoka mubushyuhe bwinshi, ntabwo rero hakenewe intambwe itandukanye yo gukora.
Ibigize
| Ibigize | HC5006A-01 | HC5006A-02 | HC5006A-03 |
Bst ADN polymerase V2 (Glycerol idafite) (8U / μL) | 0.2 mL | 1 mL | 10 mL |
| 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 |
Porogaramu
1.LAMP isothermal amplification
2.ADN umurongo umwe wimurwa
3.Urwego rwo hejuru rwa GC
4.Urutonde rwa ADN urwego rwa nanogram.
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 25 ya dNTP mubintu bitangirika aside muminota 30 kuri 65 ° C.
Kugenzura ubuziranenge
1.Isuzuma rya poroteyine (SDS-PAGE):Ubuziranenge bwa Bst ADN polymerase V2 ni ≥99% bigenwa nisesengura rya SDS-PAGE ukoresheje Coomassie Blue detection.
2.EndonucleaseIgikorwa:Inkubasi ya 50 μL reaction irimo byibura 8 U ya Bst ADN polymerase V2 hamwe na 1 μg λDNA kumasaha 16 kuri 37 ℃ bivamo nta kwangirika kugaragara nkuko byagenwe.
3.Exonuclease Igikorwa:Inkubasi ya 50 μL reaction irimo byibura 8 U ya Bst ADN polymerase V2 hamwe na 1 μg λ -Hind Ⅲ igogora ADN amasaha 16 kuri 37 ℃ bivamo nta kwangirika kugaragara nkuko byagenwe.
4.Igikorwa cya Nickase:Inkubasi ya 50 μL reaction irimo byibura 8 U ya Bst ADN polymerase V2 hamwe na 1 μg pBR322 ADN kumasaha 16 kuri 37 ° C bivamo nta kwangirika kugaragara nkuko byagenwe.
5.Igikorwa cya RNase:Inkubasi ya 50 μL reaction irimo byibura 8 U ya Bst ADN polymerase V2 hamwe na 1,6 μg MS2 RNA kumasaha 16 kuri 37 ° C bivamo nta kwangirika kugaragara nkuko byagenwe.
6.E. coliADN:120 U ya Bst ADN polymerase V2 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.
Amatara
| Ibigize | 25μL |
| 10 × HC Bst V2 Buffer | 2.5 μL |
| MgSO4 (100mM) | 1.5 μL |
| DNTPs (10mM buri umwe) | 3.5 μL |
| SYTO ™ 16 Icyatsi (25 ×)a | 1.0 μL |
| Kuvanga primerb | 6 μL |
| Bst ADN Polymerase V2 (Glycerol idafite) (8 U / uL) | 1 μL |
| Inyandikorugero | × μL |
| ddH₂O | Kugera kuri 25 μL |
Inyandiko:
1) a.SYTOTM 16 Icyatsi (25 ×): Ukurikije ubushakashatsi bukenewe, andi marangi arashobora gukoreshwa nkibisimburwa;
2) b.Kuvanga primer: byabonetse mukuvanga 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 nibindi bitabo.
Imyitwarire n'imiterere
1 × HC Bst V2 Buffer, ubushyuhe bwa incubation buri hagati ya 60 ° C na 65 ° C.
Gushyushya
80 ° C, 20min
