Rnase Kāohi
ʻO Murine RNase inhibitor kahi mea hoʻopaneʻe murine RNase hou i hōʻike ʻia a hoʻomaʻemaʻe ʻia mai E.coli.Hoʻopaʻa ia iā RNase A, B a i ʻole C ma kahi ratio 1: 1 ma o ka hoʻopaʻa ʻana non-covalent, no laila ke keʻakeʻa nei i ka hana o nā enzyme ʻekolu a pale i ka RNA mai ka hoʻohaʻahaʻa ʻana.Eia nō naʻe, ʻaʻole kūpono ia i ka RNase 1, RNase T1, S1 Nuclease, RNase H a i ʻole RNase mai Aspergillus.Ua hoʻāʻo ʻia ʻo Murine RNase inhibitor e RT-PCR, RT-qPCR a me IVT mRNA, a ua kūpono me nā ʻano transcriptases Reverse pāʻoihana, DNA polymerases a me RNA polymerases.
Ke hoʻohālikelike ʻia me nā mea hoʻopaneʻe RNase kanaka, ʻaʻole i loaʻa i ka inhibitor RNase murine ʻelua mau cysteine i koʻikoʻi loa i ka oxidation e hoʻoulu ai i ka inactivation o ka inhibitor.ʻO ia ka mea e paʻa ai i nā haʻahaʻa haʻahaʻa o DTT (emi ma mua o 1 mM).He kūpono kēia hiʻohiʻona no ka hoʻohana ʻana i nā hopena kahi i kū ʻole ai ka nui o ka DTT i ka hopena (e laʻa me RT-PCR Real-time).
Application
Hiki ke hoʻohana nui ʻia kēia huahana i kēlā me kēia hoʻokolohua kahi hiki ke hoʻopili ʻia ʻo RNase e pale aku i ka hōʻino ʻana o RNA, e like me:
1.Synthesis o ke kaula mua o cDNA, RT-PCR, RT-qPCR, etc.
2.Mālama i ka RNA mai ka hoʻohaʻahaʻa ʻana i ka wā o ka transcription/unuhi in vitro (e laʻa, in vitro viral replication system).
3.Ka pale ʻana i ka hana RNase i ka wā o ka hoʻokaʻawale ʻana a me ka hoʻomaʻemaʻe RNA.
Nā Kūlana Waihona
Hiki ke mālama ʻia ka huahana ma -25～- 15 ℃, kūpono no 2 mau makahiki.
Paʻa mālama
50 mM KCl, 20 mM HEPES-KOH (pH 7.6, 25 ℃), 8 mM DTT a me 50% glycerol.
Wehewehe ʻāpana
ʻO ka nui o ka murine RNase inhibitor i koi ʻia e kāohi i ka hana o 5ng o ribonuclease A e 50% i wehewehe ʻia he hoʻokahi ʻāpana (U).
ʻO ke kaumaha molekala o ka protein
He 50 kDa ka paona mole o ka murine RNase inhibitor.
Ka hoomalu maikai
Exonuclease Hana:
40 U o ka murine RNase inhibitor me 1 μg λ -Hind III digest DNA ma 37 ℃ no 16 mau hola ʻaʻole ia e hoʻohaʻahaʻa e like me ka mea i hoʻoholo ʻia e ka electrophoresis gel agarose.
Endonuclease hana:
40 U o ka murine RNase inhibitor me 1μg λ DNA ma 37 ℃ no 16 mau hola ʻaʻole e hāʻawi i ka degradation e like me ka hoʻoholo ʻana e ka electrophoresis gel agarose.
Nicking Hana:
40U o ka murine RNase inhibitor me 1μg pBR322 ma 37 ℃ no 16 mau hola ʻaʻole e hāʻawi i ka degradation e like me ka hoʻoholo ʻana e ka electrophoresis gel agarose.
RNase Ka hana:
ʻO 40U o murine RNase inhibitor me 1.6μ g MS2 RNA no 4h ma 37 ℃ ʻaʻole e hāʻawi i ka degradation e like me ka hoʻoholo ʻana e ka electrophoresis gel agarose.
E.coli DNA:
Hoʻopili ʻia ʻo 40 U o murine RNase inhibitor no ka loaʻa ʻana o E. coli genomic DNA e hoʻohana ana i TaqMan qPCR me nā primers kikoʻī no ka E. coli 16S rRNA locus.He ≤ 0.1 pg/40 U ka E. coli genomic DNA contamination.
Notes
1. ʻO ka oscillation ikaika a i ʻole ka hoʻoulu ʻana e alakaʻi i ka enzyme inactivation.
2. ʻO ka nui o ka mahana wela o kēia mea paʻa he 25-55 ℃, a ua hoʻopau ʻia ma 65 ℃ a ma luna.
3.ʻAʻole i kāohiʻia nā hana a RNase H, RNase 1 a me RNase T1 e ka mea hoʻopiʻi RNase murine.
4. Ua loaʻa ka inhibition o ka hana RNase ma kahi ākea o ka pH (pH 5-9 nā mea hana āpau), a ʻike ʻia ka hana kiʻekiʻe loa ma ka pH 7-8.
5. No ka mālama mau ʻana o nā ribonucleases i ka hana ma lalo o nā kūlana denaturing, pono e mālama i ka pale ʻana i ka denaturing RNase Inhibitor mole i paʻakikī me kahi ribonuclease.No ka pale ʻana i ka hoʻokuʻu ʻana o ka ribonuclease ikaika, pono e pale ʻia nā mahana i ʻoi aku ma mua o 50 °C a me nā kiʻekiʻe kiʻekiʻe o ka urea a i ʻole nā mea denaturing ʻē aʻe.
