I-TGuide S96 Igazi / Iseli / Izicubu ze-RNA Kit

I-A-1 Cell lysis noma i-homogenization ayanele

- Nciphisa ukusetshenziswa kwesampula, khulisa inani le-lysis buffer, khulisa i-homogenization nesikhathi se-lysis.

Inani elingu-A-2 lesampuli likhulu kakhulu

---- Yehlisa inani lesampula elisetshenzisiwe noma wandise inani le-lysis buffer.

I-A-1 Inganele i-lysis yeseli noma i-homogenization

- Nciphisa ukusetshenziswa kwesampula, khulisa inani le-lysis buffer, khulisa i-homogenization nesikhathi se-lysis.

Inani elingu-A-2 lesampuli likhulu kakhulu

---- Sicela ubheke amandla wokucubungula aphezulu.

I-A-3 RNA ayikhishwa ngokuphelele kukholamu

---- Ngemuva kokungeza amanzi e-RNase-Free, yiyeke imizuzu embalwa ngaphambi kwe-centrifuging.

I-A-4 Ethanol ku-eluent

---- Ngemuva kokuhlanza, i-centrifuge futhi bese ususa i-buffer yokuwasha ngangokunokwenzeka.

I-A-5 Cell culture medium ayisuswanga ngokuphelele

- Lapho uqoqa amaseli, sicela uqiniseke ukuthi ususa okuphakathi kwesiko ngangokunokwenzeka.

A-6 Amaseli agcinwe ku-RNAstore awasebenzi kahle nge-centrifuged

---- Ukuxinana kwe-RNAstore kukhulu kunesilinganiso esiphakathi sesiko leseli; ngakho-ke amandla e-centrifugal kufanele akhuliswe. Kuphakanyiswa ku-centrifuge ku-3000x g.

Okuqukethwe kwe-A-7 okuphansi kwe-RNA nobuningi kusampula

---- Sebenzisa isampula elihle ukuthola ukuthi isivuno esiphansi sidalwa yisampula.

A-1 Indaba ayisiyintsha

Izicubu ezintsha kufanele zigcinwe ku-nitrogen ewuketshezi ngokushesha noma ngokushesha zifakwe kwi-reagent ye-RNAstore ukuqinisekisa umphumela wokukhishwa.

Inani elingu-A-2 lesampuli likhulu kakhulu

---- Yehlisa inani lesampula.

I-A-3 RNase contamination

- Yize i-buffer enikezwe kukhithi ingenayo i-RNase, kulula ukungcolisa i-RNase ngesikhathi senqubo yokukhipha futhi kufanele iphathwe ngokunakekela.

Ukungcola kwe-A-4 Electrophoresis

---- Faka esikhundleni se-electrophoresis buffer futhi uqiniseke ukuthi izinto ezisetshenziswayo ne-Loading Buffer azinakho ukungcoliswa yi-RNase.

A-5 Kulayishwa kakhulu i-electrophoresis

---- Yehlisa inani lokulayishwa kwesampula, ukulayishwa komthombo ngamunye akufanele kudlule ku-2 μg.

Inani elingu-1 lesampuli likhulu kakhulu

---- Yehlisa inani lesampula.

A-2 Amanye amasampula anokuqukethwe okuphezulu kwe-DNA futhi angalashwa nge-DNase.

---- Enza ukwelashwa kwe-RNase-Free DNase kusixazululo esitholakele se-RNA, futhi i-RNA ingasetshenziswa ngqo ekuhlolweni okulandelayo ngemuva kokwelashwa, noma ingaqhubeka ihlanzwe ngamakhithi wokuhlanzwa we-RNA.

Okwezingilazi, kubhakwe ku-150 ° C ngehora elingu-4. Kwiziqukathi zepulasitiki, ucwiliswe ku-0.5 M NaOH ngemizuzu eyi-10, bese ugeza kahle ngamanzi angenawo i-RNase bese uvala inzalo ukususa i-RNase ngokuphelele. Ama-reagents noma izixazululo ezisetshenziswe ekuhlolweni, ikakhulukazi amanzi, kumele zingabi ne-RNase. Sebenzisa amanzi angenawo ama-RNase kuwo wonke amalungiselelo e-reagent (engeza amanzi ebhodleleni lengilazi elihlanzekile, engeza i-DEPC ekugxileni kokugcina okungu-0.1% (V / V), unyakazise ubusuku kanye ne-autoclave).