RNase ishoko rinonzwisa tsitsi iro vadzidzi vazhinji vanowanzo itisa maRNA ekubvisa zviedzo havadi kunzwa.Yakanyatso pfuti, iyo RNA iyo yakazoburitswa ine muchetura reagents phenol uye chloroform yakaderedzwa.Handina kuyananiswa!!!Nhasi, ngatitarisei kwakabva Rnase yakakurumbira.

Ribonuclease (RNase), kana RNase, iri nuclease inogona hydrolyze RNA kuita mamorekuru madiki.RNase, sepuroteni yemorekuru diki, yakagadzikana zvisingaite .Yakajairwa tembiricha yepamusoro uye yakanyanya kudzvanywa steam sterilization uye mapuroteni inhibitors haagone kuimisa zvachose.Kugadzikana kweRNase kunyanya kunobva kune disulfide zvisungo muchimiro.Semuenzaniso, iyo inowanzoshandiswa RNase yebovine pancreas ine 124 amino acids chete, asi ine 4 disulfide bonds.Sulfur bond uye disulfide bond endow RNase ine yakanakisa kupisa kugadzikana.Mukuwedzera, rnase ine huremu hudiki hwemamorekuru uye inogona kukurumidza kudzorera kuwirirana kwayo kwepakutanga muzviitiko zvakawanda.

Pamusoro pekugadzikana zvakanyanya,RNases anowanikwa kwose kwose murabhoritari .RNase inzira yekudzivirira zvipenyu.Kune sero, exogenous RNA inowanzouraya.Kana ichienzaniswa neiyo exogenous DNA, exogenous RNA inowanzova nengozi.RNA inonyorerwa nekududzirwa nerufaro, saka zvinenge zvipenyu zvese zvakashanduka maRNase kudzivirira kubva mukupindwa kweRNA yekunze.Naizvozvo, masero ebhakitiriya anorimwa murabhoritari uye iwe unobvisa RNA unoburitsa kunhuhwirira kweRNase.Mvura yemuviri wemunhu (mate, misodzi, nezvimwewo) ine RNase yakawanda, saka usachema kana RNA yaderedzwa.Paunochema zvakanyanya, ndiko kuwedzera kuderera kweRNA!!Hanzvadzi Daiyu haina kukodzera kubviswa kweRNA!

Pamusoro pezvo, ganda rako rakapfava rine zvakawanda zveRNase, uye mamakisi, mapipette, magonhi efiriji, uye zvibato zvegonhi zvakabatwa neganda zvakare zvine RNase.

Nekumhanya-mhanya kwakawanda, ngatitarisei maitiro ekuita neRNases.

Chinhu chekutanga chinofungwa nemunhu wese kana achibvisa RNADEPC(diethyl pyrocarbonate).DEPC inonyanya kudonhedza puroteni nekubatanidza nemidazole mhete yeRNase inoshanda boka histidine, nokudaro ichivharira basa re enzyme.0.1% DEPC inogona kuve nekubvisa zvirinani paRnase, asi isu tinofanirwa kutarisisa kuti DEPC inozivikanwa carcinogen, saka kutarisisa kwakanyanya kunofanirwa kubhadharwa kana uchiishandisa.

Kune RNase, isu tinofanirwa kutanga kubva pamativi maviri,yekutanga ndeye kurambidza kuita kwe endogenous RNase

Zvakajairwa RNA kuburitsa reagents senge guanidine isothiocyanate uye DTT iri muTrizol inogona kuvhura disulfide bond yeRNase, asi pachine mamwe maRNase, kunyanya mumasamples ematishu, saka teerera kune yakaderera tembiricha.

1.Kurumidza kunyudza sampuli yetishu mumvura ine nitrogen mushure mekuibvisa, kana mune yekutengesa RNA kuchengetedza mhinduro.

2.Mushure mekubvisa iyo RNA yesero sampuli, wedzera kune lysis mhinduro uye lyse pabhokisi rechando

3. Zvakanakisisa kushandisa mvura nitrogen pakukuya apo tishu samples dziri homogenized.Paunenge uchishandisa homogenizer yemagetsi isina nitrogen yemvura, teerera kune zvizere pre-kutonhora homogenate adapta.

Yechipiri ndeye exogenous DNase

1.Iva nezvombo zvakakwana, pfeka jasi rerabhoritari, pfeka mask, uye iva nechokwadi chekupfeka magirovhosi matsva (usave nekuchengetedza zvakanyanya!! Zvinofanira kucherechedzwa kuti Trizol inoodza zvakanyanya, uye yakasimba kwazvo kuburikidza nemagirovhosi, saka usadonhedze pamaoko ako).

2.Ese anoshandiswa pipette matipi, EP machubhu, PCR machubhu nemimwe michina inofanirwa kubviswa-RNase kurapwa.Inogona kunyikwa mu 0.1% DEPC uye yobva yamanikidzwa pasi pekumanikidzwa kwakanyanya.Nyatsoteerera kuvhiyiwa mune fume hood.Vadzvinyiriri vemunharaunda vanogona kutenga zvakananga zvinodyiwa zvekubvisa ma enzymes.

PS: Rega ndikuudze nzira ine usimbe.Kunyangwe tembiricha yepamusoro uye kudzvanywa kwepamusoro hakugone kubvisa zvachose RNase, ichabvisa chikamu chikuru chayo.2 nguva dzekupisa kwepamusoro uye kudzvanywa kwakanyanya kune mhedzisiro yakanaka, uye kubviswa kweRNA hakuna mhedzisiro.

3.Doro rinogona kukanganisa mapuroteni,saka iyo RNA yekubvisa tafura inogona kudzimwa ne75% doro , uye magirovhosi anogonawo kupfapfaidzwa nedoro.

4.Iyo yekupedzisira RNA yekunyungudutsa mhinduro uye centrifuge chubhu inofanirwawo kuve de-RNase kurapwa.DEPC mvura inowanzoshandiswa RNA dissolving solution.Ngatitaurei nezve nzira chaiyo yekugadzirira yeDEPC mvura (rangarira kurongedzazve iyo yekutengesa DEPC paunoitenga)

Wedzera DEPC kune ultrapure mvura pa1: 1000, zunza zvakanaka, rega kumira usiku hwose pa 37 ° C, uye sterilize pa 121 ° C pasi pekushisa kwepamusoro uye kudzvinyirira kwemaminetsi gumi nemashanu.DEPC mvura inogona kuchengetwa pa -20 ° C mu 1ml aliquots.

Pakupedzisira, kupfupisa: tembiricha yakaderera ndiyo kiyi, zvinodyiwa zvinobatwa nemazvo, zvine zvombo zvakakwana uye zvishoma kutaura!

Zvakanaka, ndiyo nzira yanhasi.Ndinokushuvirai mese maRNA concentration uye kuchena kwamataura.Ose A260/A280 ari 2.0!!!

Zvechokwadi, kana ukashandisa akamuri tembiricha yekushanda RNA yekubvisa kit, ungasasangana nematambudziko ari pamusoro apa.

Kushanda patembiricha yekamuri, pasina kuwedzera DNase, inobvisa yakazara RNA kubva kumaseru mumaminetsi gumi nerimwe, uye inobvisa yakazara RNA kubva munyama yemhuka kana zvidyarwa mumaminetsi makumi matatu.

Kuti uwane mienzaniso yekuedza, tapota taura:overseas@foregene.com

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