RNase ke lentsoe le hlokolosi leo baithuti ba bangata ba atisang ho etsa liteko tsa ho ntša RNA ba sa batleng ho le utloa.E hlometse ka ho feletseng, RNA eo qetellong e ileng ea ntšoa ka li-reagents tse chefo haholo phenol le chloroform e ile ea senyeha.Ha kea boelana!!!Kajeno, a re hlahlobeng tšimoloho ea Rnase e tummeng.

Ribonuclease (RNase), kapa RNase, ke nuclease e ka etsang hydrolyze RNA hore e be limolek'hule tse nyane.RNase, e le protheine e nyenyane ea molek'hule, e tsitsitse ka tsela e sa tloaelehang .Mocheso o phahameng o tloaelehileng le sterilization ea mouoane oa khatello e phahameng le li-protein inhibitors ha li khone ho e etsa ka ho feletseng.Ho tsitsa ha RNase haholo-holo ho tsoa litlamong tsa disulfide mohahong.Mohlala, RNase e sebelisoang hangata ea manyeme ea likhomo e na le li-amino acid tse 124 feela, empa e na le li-bond tse 4 tsa disulfide.Sulfur bond le disulfide bond li fa RNase e nang le botsitso bo botle ba mocheso.Ho phaella moo, rnase e na le boima ba molek'hule e batlang e le nyenyane 'me e ka tsosolosa ka potlako conformation ea eona ea pele maemong a mangata.

Ho phaella tabeng ea ho tsitsa ka ho fetisisa,Li-RNase li fumaneha hohle ka laboratoring .RNase ke mokhoa oa ts'ireletso ea likokoana-hloko.Bakeng sa sele, RNA ea kantle hangata e ea bolaea.Ha ho bapisoa le DNA ea kantle, RNA ea kantle hangata e kotsi ho feta.RNA e ngotsoe ka thabo le ho fetoleloa, kahoo hoo e batlang e le lintho tsohle tse phelang li fetohile RNase ho itšireletsa khahlanong le tlhaselo ea RNA e sa tloaelehang.Ka hona, lisele tsa baktheria tse lenngoeng ka laboratoring 'me uena u ntša RNA u ntša monko oa RNase.Mekelikeli ea 'mele oa motho (mathe, likhapha, joalo-joalo) a na le RNase e ngata, kahoo u se ke ua lla ha RNA e senyeha.Ha u ntse u lla, ke moo ho senyeha ho hoholo ha RNA!!Khaitseli Daiyu ha a tšoanelehe bakeng sa ho ntšoa ha RNA!

Ho feta moo, letlalo la hao le bonojoana le boetse le na le RNase e ngata, ’me li-marker, li-pipette, mamati a sehatsetsing, le matsoara a mamati a anngoeng ke letlalo le tsona li na le RNase.

Ka ho rarahana hakana, ha re shebeng mokhoa oa ho sebetsana le RNases.

Ntho ea pele eo motho e mong le e mong a nahanang ka eona ha a tlosa RNA keDEPC(diethyl pyrocarbonate).DEPC haholo-holo denatures protheine ka ho kopanya le imidazole reng ea RNase mafolofolo sehlopha histidine, ka tsela eo ho thibela mosebetsi oa enzyme.0.1% DEPC e ka ba le phello e ntle ea ho tlosa Rnase, empa re lokela ho ela hloko hore DEPC ke carcinogen e tsebahalang, kahoo tlhokomelo e khethehileng e lokela ho lefshoa ha e sebelisoa.

Bakeng sa RNase, re hloka ho qala ka likarolo tse peli,ea pele ke ho thibela tšebetso ea endogenous RNase

Li-reagents tse tloaelehileng tsa RNA tse kang guanidine isothiocyanate le DTT tse fumanehang ho Trizol li ka bula tlamo ea disulfide ea RNase, empa ho ntse ho e-na le li-RNase tse ling, haholo-holo lisampole tsa lisele, kahoo ela hloko mocheso o tlaase.

1.Hang-hang qoelisa sampole ea linama ka har'a metsi a naetrojene ka mor'a ho e ntša, kapa tharollong ea ho boloka RNA ea khoebo.

2.Ka mor'a ho ntša RNA ea sampuli ea sele, e kenye ka tharollo ea lysis 'me u e tšele ka lebokoseng la leqhoa

3. Ho molemo ho sebelisa naetrojene ea metsi bakeng sa ho sila ha disampole tsa dithishu di homogenized.Ha u sebelisa homogenizer ea motlakase ntle le naetrojene ea metsi, ela hloko ka ho feletseng pele ho pholile adaptara ea homogenate.

Ea bobeli ke exogenous DNase

1.E-ba le lihlomo tse feletseng, apara jase ea lab, roala maske, 'me u be le bonnete ba hore u roala liatlana tse ncha (u se ke ua baballa haholo!! Ho lokela ho hlokomeloa hore Trizol e senya haholo, hape e matla haholo ka liatlana, kahoo u se ke ua rothela matsohong a hao).

2.Malebela ohle a sebelisitsoeng a pipette, li-tubes tsa EP, li-tubes tsa PCR le lisebelisoa tse ling li tlameha ho phekoloa ka de-RNase.E ka kolobisoa ka 0.1% DEPC ebe e hatelloa tlas'a khatello e phahameng.Ela hloko ts'ebetso ea mosi oa mosi.Bahatelli ba lehae ba ka reka ka kotloloho lisebelisoa tsa ho tlosa li-enzyme.

PES: E re ke u bolelle mokhoa oa botsoa.Le hoja mocheso o phahameng le khatello e phahameng li ke ke tsa tlosa RNase ka ho feletseng, e tla tlosa karolo e kholo ea eona.Linako tse 2 tsa mocheso o phahameng le khatello e phahameng li na le phello e ntle, 'me ho ntšoa ha RNA ho na le phello e fokolang.

3.Joala bo ka senya liprotheine,kahoo tafole ea ho ntša RNA e ka hlakoloa ka joala ba 75%. , 'me liatlana li ka boela tsa fafatsoa ka joala.

4.Tharollo ea ho qetela ea RNA e qhibilihisang le tube ea centrifuge le eona e lokela ho phekoloa ka de-RNase.Metsi a DEPC ke tharollo e sebelisoang hangata ea RNA.Ha re bue ka mokhoa o nepahetseng oa ho lokisa metsi a DEPC (hopola ho khutlisetsa DEPC ea khoebo ha u e reka)

Eketsa DEPC ho metsi a ultrapure ka 1: 1000, sisinya hantle, tlohela bosiu bo le bong ho 37 ° C, 'me u etse sterilize ka 121 ° C tlas'a mocheso o phahameng le khatello e phahameng ka metsotso e 15.Metsi a DEPC a ka bolokoa ho -20 ° C ka li-aliquots tsa 1ml.

Qetellong, ho akaretsa: mocheso o tlase ke senotlolo, lisebelisoa li tšoaroa hantle, li hlometse ka ho feletseng 'me li sa bue hakaalo!

Ho lokile, ke eona bakeng sa leano la kajeno.Ke u lakaletsa mahloriso le bohloeki ba RNA bohle boo u bo boletseng.Ka bobeli A260/A280 ke 2.0!!!

Ehlile, haeba u sebelisa aOpereishene ea mocheso oa kamore ea RNA e ntšoa, mohlomong ha u tobane le mathata a ka holimo.

Ts'ebetso ka mocheso oa kamore, ntle le ho eketsa DNase, e ntša RNA eohle ho tsoa liseleng ka metsotso e 11, 'me e ntša RNA e felletseng ho tsoa liseleng tsa liphoofolo kapa limela ka metsotso e 30.

Bakeng sa mehlala ea liteko, ka kopo ikopanye le:overseas@foregene.com

Lihlahisoa tse Amanang:

https://www.foreivd.com/cell-total-rna-isolation-kit-product/

https://www.foreivd.com/animal-total-rna-isolation-kit-product/

https://www.foreivd.com/plant-total-rna/