RNase bụ okwu nwere mmetụta nke ọtụtụ ụmụ akwụkwọ na-emekarị nnwale mmịpụta RNA achọghị ịnụ.Ejiri ngwa agha zuru oke, RNA nke emechara wepụta ya na reagents phenol na chloroform na-egbu egbu mebiri emebi.Anaghị m emekọrịta!!!Taa, ka anyị leba anya na mmalite nke Rnase a ma ama.

Ribonuclease (RNase), ma ọ bụ RNase, bụ nuclease nke nwere ike ime ka RNA banye n'ime obere ụmụ irighiri ihe.RNase, dị ka obere protein molecule, kwụsiri ike nke ukwuu .Okpomọkụ dị elu nke a na-ahụkarị na mgbochi uzuoku siri ike yana ndị na-egbochi protein enweghị ike ime ya kpamkpam.Nkwụsi ike nke RNase na-esite na njikọ disulfide na nhazi ahụ.Dịka ọmụmaatụ, RNase nke pancreas nke anụ ahụ a na-ejikarị eme ihe nwere naanị amino acid 124, mana ọ nwere njikọ disulfide anọ.Njikọ Sulfur na njikọ disulfide na-enye RNase ezigbo nkwụsi ike ọkụ.Tụkwasị na nke ahụ, rnase nwere obere ihe dị arọ ma nwee ike iweghachite ọdịdị mbụ ya ngwa ngwa n'ọtụtụ ọnọdụ.

Na mgbakwunye na inwe oke kwụsiri ike,RNases dị ebe niile na ụlọ nyocha .RNase bụ usoro nchekwa ndu.Maka cell, exogenous RNA na-egbukarị egbu.E jiri ya tụnyere DNA exogenous, RNA exogenous na-adịkarị ize ndụ karị.A na-edegharị RNA nke ọma ma tụgharịa asụsụ ya, yabụ ihe fọrọ nke nta ka ọ bụrụ ihe niile dị ndụ ewepụtala RNases iji chebe onwe ya pụọ ​​​​na mbuso agha nke RNA exogenous.Ya mere, mkpụrụ ndụ nje ndị a na-akọ n'ụlọ nyocha na gị onye wepụrụ RNA na-esi ísì ụtọ nke RNase.Mmiri ahụ mmadụ (mmiri, anya mmiri, wdg) nwere nnukwu RNase, ya mere akwala ákwá mgbe RNA mebiri emebi.Ka ị na-ebe akwa, ka mbibi RNA na-akawanye njọ!!Nwanne nwanyị Daiyu adabaghị maka mmịpụta RNA!

Na mgbakwunye, akpụkpọ ahụ gị dị nro nwekwara ọtụtụ RNase, ihe nrịbama, pipettes, ọnụ ụzọ friji, na aka ọnụ ụzọ akpụkpọ ahụ metụrụ nwekwara RNase.

Site n'ịgba ọsọ dị ukwuu, ka anyị leba anya ka anyị ga-esi mesoo RNases.

Ihe mbụ onye ọ bụla na-eche mgbe ọ na-ewepụ RNA bụDEPC(diethyl pyrocarbonate).DEPC na-ewepu protein site na ijikọ ya na mgbanaka imidazole nke RNase nọ n'ọrụ histidine, si otú a na-egbochi ọrụ nke enzyme.0.1%.

Maka RNase, anyị kwesịrị ịmalite site na akụkụ abụọ,Nke mbụ bụ igbochi ọrụ nke RNAse endogenous

Mwepụta mmịpụta RNA ndị dị ka guanidine isothiocyanate na DTT dị na Trizol nwere ike imeghe njikọ nke RNase disulfide, mana a ka nwere ụfọdụ RNases, ọkachasị n'ụdị anụ ahụ, yabụ kpachara anya na obere okpomọkụ.

1.Ngwa ngwa, mikpuo ihe nlele anụ ahụ na nitrogen mmiri mmiri ka ewepụchara ya, ma ọ bụ na ngwọta nchekwa RNA azụmahịa.

2.Mgbe ị wepụsịrị RNA nke ihe nlele cell, gbakwunye ya na ngwọta lysis ma gbanye ya na igbe ice

3. Ọ kacha mma iji mmiri mmiri nitrogen maka egweri mgbe anụ ahụ samples na-homogenized.Mgbe ị na-eji homogenizer eletrik na-enweghị mmiri mmiri nitrogen, ṅaa ntị na tupu ijuo ihe nkwụnye homogenate kpamkpam.

Nke abụọ bụ exogenous DNAse

1.Jikere nke ọma, yiri uwe lab, yi ihe mkpuchi, wee hụ na ị ga-eyi uwe aka ọhụrụ (adịghị adị oke ọnụ! Ekwesịrị ịmara na Trizol bụ nnukwu ihe na-emebi emebi, ọ dịkwa ike nke ukwuu site na uwe aka, yabụ etinyela aka gị).

2.Atụmatụ pipette niile ejiri mee ihe, ọkpọkọ EP, ọkpọkọ PCR na akụrụngwa ndị ọzọ ga-emerịrị de-RNase.Enwere ike itinye ya na 0.1% DEPC wee tinye ya n'okpuru nrụgide dị elu.Lezienụ anya na ọrụ na mkpuchi uzuzu.Ndị ọchịchị aka ike n'ime obodo nwere ike zụta ngwa ahịa maka iwepu enzymes.

PS: Ka m gwa gị usoro umengwụ.Ọ bụ ezie na okpomọkụ dị elu na nrụgide dị elu enweghị ike iwepụ RNase kpamkpam, ọ ga-ewepụ nnukwu akụkụ ya.Oge 2 nke okpomọkụ dị elu na nrụgide dị elu nwere mmetụta dị mma, na mmịpụta nke RNA nwere obere mmetụta.

3.Mmanya nwere ike imebi protein,yabụ enwere ike iji mmanya 75% hichaa tebụl mmịpụta RNA , na gloves nwekwara ike fesa mmanya.

4.A ga-agwọkwa ihe ngwọta RNA ikpeazụ na centrifuge tube de-RNase.Mmiri DEPC bụ ngwọta mgbaze RNA a na-ejikarị.Ka anyị kwuo maka usoro nkwadebe ziri ezi nke mmiri DEPC (cheta ịkwaghachi DEPC azụmahịa mgbe ịzụrụ ya)

Tinye DEPC na mmiri ultrapure na 1: 1000, maa jijiji nke ọma, hapụ otu abalị na 37 Celsius C, na sterilize na 121 ° C n'okpuru nnukwu okpomọkụ na nrụgide dị elu maka nkeji 15.Enwere ike ịchekwa mmiri DEPC na -20 Celsius na 1 ml aliquots.

N'ikpeazụ, iji chịkọta: obere okpomọkụ bụ isi ihe, a na-edozi ihe oriri nke ọma, ngwa ngwa zuru oke na obere okwu!

Ọfọn, nke ahụ bụ maka atụmatụ nke taa.Asịrị m gị niile itinye uche na ịdị ọcha nke RNA ị kwuru.Abụọ A260 / A280 bụ 2.0!!!

N'ezie, ọ bụrụ na ị na-eji angwa RNA na-arụ ọrụ okpomọkụ, ị nwere ike ọ gaghị ezute nsogbu ndị dị n'elu.

Ịrụ ọrụ n'ime ụlọ okpomọkụ, na-agbakwunyeghị DNAse, na-ewepụta mkpokọta RNA na sel n'ime nkeji iri na otu, wee wepụ RNA zuru ezu na anụ ahụ ma ọ bụ osisi n'ime nkeji 30.

Maka nlele nnwale, biko kpọtụrụ:overseas@foregene.com

Ngwaahịa ndị emetụtara:

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/