Igitabo cyo gusesengura ibibazo

Isesengura rikurikira ryibibazo ushobora guhura nabyoIgiterwa cyoseRNA extraction will help you with your experiments. In addition, for other experimental or technical problems in addition to operating instructions and problem analysis, we have dedicated technical support to help you. If you have any needs, please contact us at: 028-83360257 or E-mali : Tech@foregene.com.

Inkingi izunguruka ifunze

Guhagarika inkingi zizunguruka bizatera umusaruro wa RNA kugabanuka cyangwa no kudashobora kwezwa kugirango ubone RNA, kandi ubwiza bwa RNA wabonye buzaba buke.

Isesengura rusange risanzwe:

1. Icyitegererezo nticyacitse burundu.

Gutandukanya ibyitegererezo bituzuye birashobora guhagarika ADN-Isukura Inkingi, ishobora no kugira ingaruka kumusaruro wa RNA nubwiza.Turasaba ko mugihe ukora ibice byintangarugero, gusya vuba mukigero gike cya azote yuzuye kugirango ucike ingirangingo nkurukuta rwakagari hamwe nibice by'uturemangingo bishoboka.Kubitegererezo byibihingwa bya polifenol polysaccharide, turagusaba ko ukoresha Ibimera byose RNA Isolation Kit Plus.

2.Kora ADN-Isukura Inkingi yitaruye ndengakamere, wifuze imyanda ishobora guterwa.

Pellet selile yifuzwa irashobora gufunga Inkingi ya RNA gusa mugihe cya adsorption ya RNA (reba intambwe ya 5 yuburyo, intambwe ya 6 yuburyo bwa polysaccharide polifenol).Turasaba ko hagomba kwitonderwa mugihe wifuza iyi ndengakamere kugirango wirinde imyanda ya selile.

3. Umubare wambere wicyitegererezo ni munini cyane.

Gukoresha urugero rwinshi bizavamo gutandukanya ibyitegererezo bituzuye cyangwa lysis ituzuye ya selile na Buffer PRL1 cyangwa Buffer PSL1, bikavamo inkingi yo kweza ifunze kubikorwa byo kweza.Igiterwa Igiteranyo cya RNA Isolation Kit gifite intangiriro ntarengwa ya mg 50 kuri buri kweza icyitegererezo cyakorewe.Kubitegererezo byibihingwa bya polifenol polysaccharide, turagusaba ko wagerageza Ibimera byose RNA Isolation Kit Plus.

4. Ubushyuhe bwa centrifuge buri hasi cyane.

Kwigunga kwa RNA kwose no kwezwa usibye guhagarika azote yuzuye ya tissue yintangarugero, intambwe zose zikorwa mubushyuhe bwicyumba (20-25 ° C).Centrifuges zimwe zifite ubushyuhe buke zifite ubushyuhe buri munsi ya 20 ° C, zishobora gutera inzitizi muri ADN-Isukura Inkingi na / cyangwa RNA-Inkingi gusa.Niba ibi bibaye, shyira ubushyuhe bwa centrifuge kuri 20-25 ° C hanyuma ubanze ushyushye ivangwa rya lysis hamwe na / cyangwa wongeyeho itandukanyirizo rya Ethanol ndengakamere kuri 37 ° C.

Nta RNA yakuweho cyangwa umusaruro wa RNA uri muke

Mubisanzwe hariho ibintu byinshi bigira ingaruka kumikorere yo kugarura, nka: icyitegererezo cya RNA, uburyo bwo gukora, ingano ya elution, nibindi.

Isesengura ry'impamvu zisanzwe nkuko bikurikira:

1.Ubwogero bwa ice cyangwa ubushyuhe buke (4 ° C) centrifugation yakozwe mugihe cyo kubaga.

Igitekerezo: Kora ku bushyuhe bwicyumba (15-25 ° C) mubikorwa byose, ntukore ubwogero bwa ice hamwe na centrifugation yubushyuhe buke.

       2.RNA yangiritse kubera kubika neza icyitegererezo cyangwa kubika igihe kirekire icyitegererezo.

Icyifuzo: Ingero zegeranijwe vuba zigomba gukonjeshwa vuba muri azote yuzuye, hanyuma zikabikwa kuri -80 ° C igihe kirekire, ukirinda gukonjesha no gukonjesha ingero;cyangwa uhite ushiramo ibyitegererezo muri RNA stabilisateur RNAlater igisubizo (icyitegererezo cyinyamaswa).

       3.Icyitegererezo kidahagije hamwe na lysis biganisha ku guhagarika inkingi yo kweza.

Igitekerezo: Mugihe cyo gusya ingirangingo, nyamuneka urebe neza ko tissue iri hasi bihagije, kandi uyimure vuba muri Buffer PSL1 yateguwe mbere (wemeze ko igipimo gikwiye cya β-ME cyongeweho, reba intambwe ya 1 yuburyo).

4.Umuhanga wongeyeho nabi.

Igitekerezo: Menya neza ko ddH idafite RNase₂O yatonywe hagati yo kweza inkingi ya membrane.

5.Ubunini bukwiye bwa Ethanol yuzuye ntabwo yongewe kuri Buffer PSL2 cyangwa Buffer PRW2.

Igitekerezo: Nyamuneka kurikiza amabwiriza, ongeramo ingano yukuri ya Ethanol yuzuye kuri Buffer PSL2 na Buffer PRW2 hanyuma uvange neza mbere yuko ibikoresho bikoreshwa.

6. Ingano yicyitegererezo ntikwiye.

Igitekerezo: Koresha mg 50 za tissue kuri 500 μl ya Buffer PSL1.Gukoresha tissue nyinshi bizagabanya ingano ya RNA yakuweho kandi isuku ya RNA yavuyemo nayo izagabanuka.Turasaba cyane ko urugero rwambere rwicyitegererezo rutagomba kurenza mg 50 kubikorwa byo gukuramo RNA.

7. Ingano ya elution idakwiye cyangwa gusiba kutuzuye.

Igitekerezo: Ingano yoroheje yinkingi yo kweza ni 50-200 μl;niba ingaruka zo gukuraho zidashimishije, birasabwa kongera umwanya mubushyuhe bwicyumba nyuma yo kongeramo ubushyuhe bwa RNase-Ubusa ddH₂O, nka 5-10min.

8.Inkingi yo kweza ifite ibisigisigi bya Ethanol nyuma yo gukaraba hamwe na Buffer PRW2.

   Igitekerezo: Niba umuyoboro wubusa ushyizwemo min min 1 kandi haracyari etanol isigaye nyuma yo koza muri Buffer PRW2, urashobora kongera igihe cya centrifugation yubusa kugeza min 2, cyangwa ugashyira inkingi yo kweza mubushyuhe bwicyumba kuminota 5 kugirango ukureho Ethanol isigaye.

9.Ibikoresho byakoreshejwe nabi.

   Icyifuzo: Kubihingwa by'icyitegererezo cya polifenolike polysaccharide, ukoresheje ibikoresho bisanzwe nka Plant Total RNA Isolation Kit ntibishobora kubona urugero rwiza rwa RNA.Turagusaba gukoresha Ikimera Cyuzuye RNA IsolationKit Plus, yagenewe byumwihariko kuburugero rwibiti bya polifenolike polysaccharide.Igikoresho cyakozwe muburyo bwo gukuramo RNA muri polifenol na polysaccharide yintangarugero.

OD260 / OD280 agaciro kari hasi

Gukuraho RNA hamwe na ddH₂O kandi ikoreshwa kubisomwa bya spekitifotometero ibisubizo bya OD260 / OD280.Turasaba gukoresha mM 10 Tris-HCl, pH 7.5 (kuruta ddH ya RNase₂O kugirango uhindure RNA) kugirango ubone indangagaciro za OD260 / OD280, reba "RNA Kwibanda no Kweza" kurupapuro rwa 19.

RNA yatunganijwe yangiritse

Ubwiza bwa RNA isukuye bujyanye nibintu nko kubungabunga icyitegererezo, kwanduza RNase, no gukoreshwa.

Isesengura ry'impamvu zisanzwe:

1.Icyitegererezo nticyabitswe mugihe cyo gukusanya.

    Icyifuzo: Niba ingero za tissue zidakoreshejwe mugihe cyo gukusanya, nyamuneka uzibike muri azote yuzuye mumazi yubushyuhe buke ako kanya cyangwa uyohereze kuri -80 ° C kugirango ubike igihe kirekire nyuma yo gukonjeshwa vuba muri azote yuzuye, cyangwa uhite wibiza ibyitegererezo muri RNA stabilizer RNAlater igisubizo (icyitegererezo cyinyamaswa).Kubikuramo RNA, gerageza gukoresha ingero za tissue nshya zegeranijwe.

2.Gusubiramo kenshi no gukonjesha ingero za tissue.

   Icyifuzo: Iyo ubitse ingero za tissue, nibyiza kubicamo uduce duto kugirango tubungabunge, hanyuma ugakuramo igice cyabyo mugihe ubikoresheje kugirango wirinde kwangirika kwa RNA guterwa no gukonjesha no gukonjesha ingero.

3.RNase yatangijwe mucyumba cyo gukoreramo cyangwa ntabwo yambarwa na gants imwe, masike, nibindi.

   Icyifuzo: Ubushakashatsi bwo gukuramo RNA bukorwa neza mubikorwa bitandukanye bya RNA, kandi ameza ya laboratoire agomba gusukurwa mbere yubushakashatsi, kandi uturindantoki twajugunywe hamwe na masike bigomba kwambarwa mugihe cyibigeragezo kugirango twirinde kwangirika kwa RNA guterwa no kwinjiza RNase ku rugero runini.

4.Ragent yanduye na RNase mugihe cyo kuyikoresha.

   Icyifuzo: Simbuza urukurikirane rushya rwibimera byose RNA ikuramo ibikoresho kubushakashatsi bujyanye.

5.Icyuma cya centrifuge hamwe na pipette inama zikoreshwa muri RNA manipulation zanduye na RNase.

Igitekerezo: Menya neza ko imiyoboro ya centrifuge, inama za pipeti, imiyoboro, nibindi bikoreshwa mugukuramo RNA byose ni RNase-Ubuntu.

Imfashanyigisho:

Gutera Igitabo Cyuzuye cya RNA Kwigunga Igitabo