Rnase A
Ko te Ribonuclease A(RNaseA) he polypeptide kotahi-waa kei roto e 4 nga here disulfide me te taumaha ngota o te 13.7 kDa.Ko te RNase Ais he endoribonuclease e tino whakaheke ana i te RNA whenu kotahi ki nga toenga C me U.Ko te tikanga, ka mohio te wehenga ki te here phosphodiester i hangaia e te 5'-ribose o te nucleotide me te roopu phosphate i runga i te 3'-ribose o te pirimidine nucleotide e tata ana, na reira ka hydrolyzed te 2,3'-Cyclic phosphates ki te 3 e rite ana. 'Nucleoside phosphates (hei tauira, pG-pG-pC-pA-pG ka wehea e RNase A hei whakaputa pG-pG-pCp me A-PG).Ko te RNase A te mea tino kaha ki te wawahi i te RNA whenu kotahi.Ko 1- 100 μG/mL te kukū mahi e taunaki ana, he hototahi ki nga momo punaha tauhohenga.Ka taea te whakamahi i te kukū iti o te tote (0-100 mM NaCl) ki te tapahi i te RNA tautahi, te RNA taurua, me nga mekameka RNA i hangaia e te RNA-DNA hybridization.
Heoi, i te kukū o te tote nui (≥0.3 M), ka wehe motuhake a RNase A i te RNA whenu kotahi.
Ko te RNase A te nuinga o te whakamahi hei tango i te RNA i te wa e whakarite ana te DNA plasmid, te DNA genomic ranei.Ahakoa kei te kaha te DNase i te wa o te whakaritenga ka taea te pa ki te tauhohenga.Ko te tikanga tuku iho mo te koropupu i roto i te pati wai ka taea te whakamahi hei whakakore i te mahi DNase.Ko tenei hua kaore he DNase me te protease, kaore e hiahiatia he maimoatanga wera i mua i te whakamahinga.Hei taapiri, ka taea hoki te whakamahi i tenei hua ki nga whakamatautau koiora irapoi penei i te tātari tiaki RNase me te tātari raupapa RNA.
Nga tikanga rokiroki
Me penapena te hua ki te -25 ℃~- 15 ℃ mo te 2 tau.
Whakatakotoranga
| Te ahua | Paura |
| Te nui | 100mg / 1g |
| Momo hua | RNase A |
Tohutohu
Koinei tetahi o nga tikanga noa mo te whakarite RNase A otinga rokiroki.Ka taea hoki te whakaritena etahi atu tikanga e ai ki nga tikanga tuku iho i roto i te taiwhanga, tuhinga tohutoro ranei (penei irewa tika i roto i te 10 mM Tris-HCl, pH 7.5, Tris-NaCl wairewa ranei)
1. Whakamahia te 10 mM konutai acetate (pH 5.2) hei whakarite i te 10 mg/mL o te otinga rokiroki RNase A.
2. Whakawera i te 100 ℃ mo te 15 min.
3. Matao ki te pāmahana rūma, tāpirihia te 1/10 rōrahi o te 1 M Tris-HCl (pH 7.4), whakatikahia tōna pH ki te 7.4 (motauira, tāpiri 500 mL o 10 mg/mL RNase otinga rokiroki 1 M Tris-HCl, pH7.4).
4. Kua whakaemihia ki te -20 ℃ mo te rokiroki tio, ka taea te pumau mo te 2 tau.
[Tuhipoka]:
I te wa e kohuatia ana te otinga RNaseA i raro i nga tikanga kore, ka puta te rerenga RNase;Kohuatia ki te iti ake o te pH, a, ki te mea he ua, ka taea te kite, tera pea na te noho o te poke o te pūmua.Mena ka kitea te parataiao i muri i te kohua, ka taea te tango i nga poke ma te tere o te centrifugation (13000rpm), katahi ka whakakiia mo te rokiroki mīti.
Notes
Tena koa mau nga PPE e tika ana, penei i te koti taiwhanga me nga karapu, hei whakarite i to hauora me to haumaru.
