Iiseli ze-TGuide / iTissue / iRNA Kit

I-A-1 Cell lysis okanye i-homogenization ayonelanga

Ukunciphisa ukusetyenziswa kwesampula, ukwandisa inani le-buffer ye-lysis, ukwandisa i-homogenization kunye nexesha le-lysis.

Isixa sesampulu esingu-A-2 sikhulu kakhulu

---- Ukunciphisa inani lesampuli esetyenzisiweyo okanye ukwandisa inani le-lysis buffer.

A-1 I-lysis yeseli engonelanga okanye i-homogenization

Ukunciphisa ukusetyenziswa kwesampula, ukwandisa inani le-buffer ye-lysis, ukwandisa i-homogenization kunye nexesha le-lysis.

Isixa sesampulu esingu-A-2 sikhulu kakhulu

- Nceda ubhekise kubungakanani bokusebenza.

I-A-3 RNA ayikhutshiwe ngokupheleleyo kwikholamu

--Ngasemva kokongeza amanzi asimahla e-RNase, yishiye imizuzu embalwa ngaphambi kokufaka isenti.

I-A-4 Ethanol elufeni

- Emva kokucoca, icentrifuge kwakhona kwaye ususe isikhuseli sokuhlamba kangangoko.

I-A-5 yesiko lenkcubeko yeseli ayisuswanga ngokupheleleyo

- Xa uqokelela iiseli, nceda uqiniseke ukuba ususa indawo yenkcubeko kangangoko kunokwenzeka.

A-6 Iiseli ezigcinwe kwiRNAstore azisebenzi kakuhle kwisenti

--Uxinano lweRNAstore lukhulu kunophakathi ophakathi kwenkcubeko yeseli; ke amandla e-centrifugal kufuneka anyuswe. Kuyacetyiswa kwi-centrifuge kwi-3000x g.

Umxholo we-A-7 Low RNA kunye nobuninzi kwisampulu

--Sebenzisa isampulu elungileyo ukumisela ukuba ngaba isivuno esisezantsi sibangelwa sisampulu.

A-1 Imathiriyeli ayisiyonto intsha

Izicubu ezintsha kufuneka zigcinwe kwi-nitrojeni engamanzi kwangoko okanye kwangoko ibekwe kwi-RNAstore reagent ukuqinisekisa ukusebenza kokukhutshwa.

Isixa sesampulu esingu-A-2 sikhulu kakhulu

---- Ukunciphisa inani lesampula.

A-3 RNase ukungcolan

- Nangona i-buffer ebonelelwe kwikhithi ingenayo i-RNase, kulula ukungcolisa i-RNase ngexesha lenkqubo yokukhupha kwaye kufuneka iphathwe ngononophelo.

Ungcoliseko lwe-4-Electrophoresis

---- Faka endaweni ye-electrophoresis buffer kwaye uqiniseke ukuba izinto ezisetyenziswayo kunye nokuLayisha iBuffer zikhululekile kungcoliseko lweRNase.

A-5 Ukulayisha kakhulu i-electrophoresis

---- Ukunciphisa inani lokulayisha isampula, ukulayishwa kwequla ngalinye akufuneki kudlule kwi-2 μg.

A-1 Isixa sesampulu sikhulu kakhulu

---- Ukunciphisa inani lesampula.

A-2 Ezinye iisampulu zinomxholo ophezulu we-DNA kwaye zinokunyangwa nge-DNase.

- Yenza unyango lwe-RNase-Simahla ye-DNase kwisisombululo esifunyenweyo se-RNA, kwaye i-RNA inokusetyenziselwa ngokuthe ngqo kuvavanyo olulandelayo emva konyango, okanye inokucocwa ngakumbi ziikhithi zokucocwa zeRNA.

Ngeiglasi, kubhakwe kwi-150 ° C nge-4 h. Kwizikhongozeli zeplastiki, zibhaptizwe kwi-0.5 M NaOH kangange-10 min, emva koko zahlanjwa kakuhle ngamanzi angenasimahla e-RNase kwaye emva koko zibulale ukuze ususe i-RNase ngokupheleleyo. Izinto ezisetyenziswayo okanye isisombululo esisetyenzisiweyo kulingo, ngakumbi amanzi, kufuneka simahla kwi-RNase. Sebenzisa amanzi asimahla e-RNase kuwo onke amalungiselelo e-reagent (yongeza amanzi kwibhotile yeglasi ecocekileyo, yongeza i-DEPC kuxinzelelo lokugqibela lwe-0.1% (V / V), gungqisa ubusuku kunye ne-autoclave).