RNase yog ib lo lus rhiab heev uas ntau tus tub ntxhais kawm uas feem ntau ua RNA rho tawm kev sim tsis xav hnov.Tag nrho cov riam phom, RNA uas thaum kawg tau muab rho tawm nrog cov tshuaj lom phenol thiab chloroform yog degraded.Kuv tsis sib haum!!!Niaj hnub no, cia peb saib lub hauv paus chiv keeb ntawm Rnase nto moo.

Ribonuclease (RNase), los yog RNase, yog ib tug nuclease uas muaj peev xwm hydrolyze RNA rau hauv me me molecules.RNase, raws li ib tug me me molecule protein, yog txawv txawv .Cov pa kub thiab kub siab ua kom tsis muaj menyuam thiab cov protein inhibitors tsis tuaj yeem ua kom tsis muaj zog.Kev ruaj ntseg ntawm RNase feem ntau yog los ntawm disulfide bonds hauv cov qauv.Piv txwv li, feem ntau siv RNase ntawm bovine pancreas tsuas muaj 124 amino acids, tab sis muaj 4 disulfide bonds.Sulfur bond thiab disulfide bond endow RNase nrog zoo thermal stability.Tsis tas li ntawd, rnase muaj qhov hnyav molecular me me thiab tuaj yeem rov qab kho nws qhov kev sib haum xeeb hauv ntau qhov xwm txheej.

Ntxiv nrog rau kev ruaj khov heev,RNases yog ubiquitous nyob rau hauv lub laboratory .RNase yog ib qho kev tiv thaiv kab mob lom.Rau ib lub cell, exogenous RNA feem ntau ua rau tuag taus.Piv nrog exogenous DNA, exogenous RNA feem ntau txaus ntshai.RNA zoo siab thiab txhais lus, yog li yuav luag txhua yam kab mob tau hloov kho RNases los tiv thaiv kev cuam tshuam ntawm exogenous RNA.Yog li ntawd, cov kab mob ntawm cov kab mob cultivated nyob rau hauv lub laboratory thiab koj uas extract RNA exude lub aroma ntawm RNase.Tib neeg lub cev kua (qaib, kua muag, thiab lwm yam) muaj ntau ntawm RNase, yog li tsis txhob quaj thaum RNA degraded.Qhov ntau koj quaj, qhov phem RNA degradation!!Muam Daiyu tsis haum rau RNA rho tawm!

Tsis tas li ntawd, koj cov tawv nqaij ilv kuj muaj ntau RNase, thiab cov cim, pipettes, lub qhov rooj tub yees, thiab lub qhov rooj uas tau kov ntawm daim tawv nqaij kuj muaj RNase.

Nrog ntau qhov kev sib tw, cia peb saib yuav ua li cas nrog RNases.

Thawj qhov uas txhua tus xav txog thaum tshem RNA yogDEPC(Diethyl pyrocarbonate).DEPC feem ntau denatures cov protein los ntawm kev sib txuas nrog lub nplhaib imidazole ntawm RNase nquag pab pawg histidine, yog li inhibiting kev ua haujlwm ntawm enzyme.0.1% DEPC tuaj yeem muaj kev tshem tawm zoo dua ntawm Rnase, tab sis peb yuav tsum tau saib xyuas tias DEPC yog cov kab mob carcinogen, yog li yuav tsum tau them nyiaj tshwj xeeb thaum siv nws.

Rau RNase, peb yuav tsum pib los ntawm ob yam,thawj yog inhibit qhov kev ua ntawm endogenous RNase

Cov pa RNA extraction reagents xws li guanidine isothiocyanate thiab DTT muaj nyob rau hauv Trizol tuaj yeem qhib cov disulfide daim ntawv cog lus ntawm RNase, tab sis tseem muaj qee cov RNases, tshwj xeeb tshaj yog nyob rau hauv cov ntaub so ntswg kuaj, yog li them sai sai rau qhov kub thiab txias.

1.Tam sim ntawd muab cov ntaub so ntswg tso rau hauv cov kua nitrogen tom qab tshem tawm, lossis hauv kev lag luam RNA kev txuag tshuaj.

2.Tom qab rho tawm RNA ntawm cov qauv ntawm tes, ntxiv rau cov tshuaj lysis thiab lyse rau ntawm lub thawv dej khov.

3. Nws yog qhov zoo tshaj plaws los siv cov kua nitrogen rau kev sib tsoo thaum cov ntaub so ntswg kuaj yog homogenized.Thaum siv hluav taws xob homogenizer tsis muaj kua nitrogen, ua tib zoo saib ua ntej kom txias rau homogenate adapter.

Qhov thib ob yog exogenous DNase

1.Ua kom muaj tub rog, hnav lub tsho (lab), hnav lub npog ntsej muag, thiab nco ntsoov hnav ob lub hnab looj tes tshiab (tsis txhob ua kom zoo !! Nws yuav tsum tau muab sau tseg tias Trizol yog super corrosive, thiab nws tseem muaj zog heev los ntawm hnab looj tes, yog li tsis txhob poob ntawm koj txhais tes).

2.Tag nrho cov lus qhia siv pipette, EP raj, PCR hlab thiab lwm yam khoom siv yuav tsum tau kho de-RNase.Nws tuaj yeem tsau rau hauv 0.1% DEPC thiab tom qab ntawd nias rau hauv siab.Ua tib zoo saib xyuas kev ua haujlwm hauv fume hood.Cov tyrants hauv zos tuaj yeem yuav khoom siv ncaj qha rau kev tshem tawm cov enzymes.

PS: Cia kuv qhia koj ib txoj kev tub nkeeg.Txawm hais tias qhov kub thiab txias siab tsis tuaj yeem tshem tawm RNase kiag li, nws yuav tshem tawm ib feem ntawm nws.2 zaug ntawm qhov kub thiab txias siab muaj qhov zoo, thiab kev rho tawm ntawm RNA tsis tshua muaj txiaj ntsig.

3.Cawv tuaj yeem tsim cov protein,yog li lub rooj rho tawm RNA tuaj yeem so nrog 75% cawv , thiab hnab looj tes kuj tuaj yeem txau nrog cawv.

4.Qhov kawg RNA dissolving tov thiab centrifuge raj yuav tsum tau kho de-RNase.DEPC dej yog ib qho kev siv RNA dissolving tov.Cia peb tham txog txoj kev npaj kom raug ntawm DEPC dej (nco ntsoov rov ntim cov khoom lag luam DEPC thaum koj yuav nws)

Ntxiv DEPC rau cov dej ntshiab ntawm 1: 1000, co zoo, cia sawv ntsug hmo ntuj ntawm 37 ° C, thiab sterilize ntawm 121 ° C nyob rau hauv qhov kub thiab txias rau 15 feeb.DEPC dej tuaj yeem khaws cia ntawm -20 ° C hauv 1ml aliquots.

Thaum kawg, kom suav hais tias: qhov kub thiab txias yog qhov tseem ceeb, cov khoom siv tau raug tswj xyuas kom zoo, tag nrho cov riam phom thiab hais lus tsawg dua!

Zoo, uas yog nws rau hnub no lub tswv yim.Kuv xav kom koj tag nrho RNA concentration thiab purity koj hais.Ob qho A260 / A280 yog 2.0!!!

Tau kawg, yog tias koj siv achav kub ua haujlwm RNA rho tawm cov khoom siv, tej zaum koj yuav tsis ntsib cov teeb meem saum toj no.

Kev ua haujlwm ntawm chav tsev kub, tsis ntxiv DNase, rho tawm tag nrho RNA los ntawm cov hlwb hauv 11 feeb, thiab rho tag nrho RNA los ntawm cov ntaub so ntswg tsiaj lossis cov nroj tsuag hauv 30 feeb.

Rau cov qauv sim, thov hu rau:overseas@foregene.com

Yam khoom:

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/