Uracil DNA Glycosylase
Uracil-DNA Glycosylase (UNG kana UDG) iri recombinant clone yeE.coli ine molecular huremu hwe25 kDa.Iyo inokonzeresa kuburitswa kwemahara uracil kubva kuuracil-ine imwe-yakasungwa uye yakapetwa kaviri DNA, uye isingaite kurwisa RNA, uye inogona kushandiswa kudzivirira kusvibiswa kwePCR amplification zvigadzirwa.Nheyo yekuita yakavakirwa pachokwadi chekuti kana dUTP ikatsiviwa nedTTP muPCR reaction uye PCR amplification chigadzirwa chine dU mabhesi akaumbwa, iyo enzyme inogona kusarudza kutyora glycosidic bond yeU mabhesi mune imwechete-yakasungwa uye yakapetwa kaviri. DNA uye kuderedza PCR amplification chigadzirwa.
Recommended Application
Contamination Prevention Amplification
Storage Condition
-20°C kuitira kuchengetedza kwenguva refu, inofanira kusanganiswa zvakanaka isati yashandiswa, dzivisa kugaro chando-nyorova.
Storage buffer
20 mM Tris-HCl (pH 8.0) , 150 mM NaCl, 1 mM EDTA, 1 mM DTT, Stabilizer, 50% Glycerol.
Chikamu Tsanangudzo
Huwandu hwe enzyme inodiwa kuderedza 1µg ye-single-stranded DNA ine dU mabhesi muawa imwe pa37 ° C i1 unit.
Quality Control
1.SDS-PAGE electrophoretic kuchena kukuru kupfuura 98%
2.Amplification senitivity, batch-to-batch control, kugadzikana
3.Mushure mekunge 1U yeUNG yarapwa pa50 ℃ kwe2mins, iyo template ine U pazasi 103 makopi inofanira kuderedzwa zvachose uye hapana chigadzirwa chekusimudzira chinogona kugadzirwa.
4.Hapana exogenous nuclease chiitiko
Mirayiridzo
| Zvikamu | Vhoriyamu (μL) | Final concentration |
| 10 × PCR Buffer (dNTP yemahara, Mg²+mahara) | 5 | 1× |
| dUTPs (dCTP, dGTP, dATP) | - | 200 μM |
| dUTP (tsiva dTTP) | - | 200-600 μM |
| 25 mM MgCl2 | 2-8 μL | 1-4 mM |
| 5 U/μL Taq | 0.25 | 1.25 U |
| 5 U/μL UNG | 0.25 (0.1-0.5) | 0.25 U (0.1-0.5) |
| 25 × Primer Mixa | 2 | 1× |
| Template | - | ＜1μg/maitiro |
| ddH₂O | kusvika ku50 | - |
Cherechedza: a: Kana yakashandiswa qPCR/qRT-PCR, iyo fluorescent probe inofanira kuwedzerwa mune reaction system.Kazhinji, yekupedzisira primer concentration ye 0.2 μM inogona kupa zvibereko zvakanaka;kana kuita kwekuita kwakashata, iyo primer concentration inogona kugadziridzwa muhuwandu hwe 0.2-1 μM.Kazhinji, iyo probe concentration inogadziriswa muhuwandu hwe0.1-0.3 μM.Concentration gradient miedzo inogona kuitwa kuti uwane yakanakisa musanganiswa weprimer uye probe.
Notes
1.UNG enzyme inogona kushandiswa kubvisa yakasvibiswa dUTP amplification zvigadzirwa kubva kune reaction system pamberi pePCR amplification reaction, ipapo kudzivirira nhema-zvakanaka mhedzisiro nekuda kwekusvibiswa kwechigadzirwa.
2.Iyo yakakwana tembiricha yeUNG enzyme kuti ishandiswe mukupokana-kusvibiswa PCR maitiro anowanzo 50 ℃ kwe2mins;iyo inactivation mamiriro ndeye 95 ℃ ye5mins.
3.Dzivisa chando chinogara chichinyunguduka, uye usafumure kushanduka kukuru kwetembiricha.
4.Majini akasiyana kuti akwidziridzwe ane akasiyana mashandisiro e dUTP uye senitivity kune UNG enzyme, saka, kana kushandiswa kweUN system kunotungamirira kukudzikira kwekunzwa kwekuona, maitiro ekuita anofanirwa kugadziridzwa uye kugadziridzwa, kana iwe uchida rubatsiro rwehunyanzvi, ndapota taura. kambani yedu.
