FAQ's eeXirmada Go'doominta RNA Wadarta Xoolaha

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RNA lama soo saarin ama wax soo saarka RNA waa yar yahay

Inta badan waxaa jira arrimo kala duwan oo saameeya waxtarka soo kabashada, sida: muunada unugyada RNA content, habka hawlgalka, mugga elution, iwm.

1. Barafka qubeyska ama cryogenic (4°C) centrifugation ayaa la sameeyay intii lagu jiray qaliinka.

Talo soo jeedin: Ku shaqee heerkulka qolka (15-25 ° C) inta lagu jiro habka oo dhan, ha ku maydhin baraf oo ha ku daboolin heerkul hoose.

2. Ilaalinta muunad khaldan ama wakhtiga kaydinta muunada xad dhaafka ah.

Talo-soo-jeedin: Ku kaydi shay-baarka--80 °C ama ku qabooji nitrogen dareeraha ah kana fogow isticmaalka baraf-dhalashada ee soo noqnoqda;isku day inaad u isticmaasho unug cusub ama unugyo dhaqan leh soo saarista RNA.

3. Shaybaar aan ku filnayn.

Talo soo jeedin: Marka unugyadu isku mid yihiin, hubi in unuggu si ku filan isu ekaysiiyo iyo in unugyadu si ku filan u kala qaybsamaan si loo sharaxo sii daynta RNA.

4. Eluent si sax ah looguma darin.

Talo soo jeedin: Xaqiiji in RNase-free ddH₂O waxaa lagu daraa hoos udhaca dhexe ee xuubka tiirka sifaynta.

5. Mugga saxda ah ee ethanol buuxa laguma darin Buffer RL2 ama Buffer RW2.

Talo soo jeedin: Raac tilmaamaha, ku dar mugga saxda ah ee ethanol buuxa ee Buffer RL2 iyo Buffer RW2 oo si fiican isku qas ka hor intaadan isticmaalin qalabka.

6. Qiyaasta muunadda unugyada ma habboona.

Talo soo jeedin: Isticmaal 10-20 mg oo unug ah ama (1-5) × 10⁶unug kasta 500 μl baffer RL1, maadaama isticmaalka nudaha xad-dhaafka ah ay keeni karto soo saarista RNA oo yaraada.

7. Mugga sare-u-qaadista oo aan sax ahayn ama muuqaal aan dhammaystirnayn.

Talo soo jeedin: Mugga sare ee tiirka nadiifinta waa 50-200 μl;Haddii saamaynta sare-u-qaadista aysan ku qanacsanayn, waxaa lagu talinayaa in la kordhiyo waqtiga meelaynta heerkulka qolka ka dib marka lagu daro RNase-free ddH preheated₂O, tusaale ahaan 5-10 daqiiqo

8.Tiirka nadiifinta ayaa leh hadhaaga ethanol ka dib markii la dhaqo Buffer RW2.

Talo soo jeedin: Haddii ay jirto hadhaaga ethanol ka dib markii la dhaqo Buffer RW2, centrifugation tube madhan 1min, wakhtiga hawlgalka centrifugation tuubada madhan waxaa la kordhin karaa ilaa 2min, ama tiirka sifaynta waxaa la dhigi karaa heerkulka qolka 5 min si ku filan looga saaro ethanol hadhaaga ah.

RNA la safeeyey ayaa hoos u dhacay

Tayada RNA la safeeyey waxay la xiriirtaa arrimo ay ka mid yihiin ilaalinta shaybaarka, faddaraynta RNase, iyo khalkhalgelinta, iwm.

1. Tijaabada unugyada lama hayo wakhtigeeda.

Talo soo jeedin: Haddii shaybaarada unugyada ama unugyada aan loo isticmaalin waqti ku habboon ka dib ururinta, isla markiiba cryopreserve at -80 °C ama nitrogen dareere ah.Si aad u soo saarto RNA, isticmaal unug cusub oo la qaaday ama muunad unug mar kasta oo ay suurtogal tahay.

2. Baraf-dhalaalitaan soo noqnoqda ee shaybaarada unugyada.

Talo soo jeedin: Marka la kaydinayo shaybaarka unugyada, waxa fiican in la jarjaro qaybo yaryar si loo xafido, oo laga saaro mid ka mid ah qaybaha marka la isticmaalayo si looga fogaado baraf-dhalashada soo noqnoqda ee muunada iyo hoos u dhaca RNA.

3. RNase waa la soo bandhigay ama aan la xidhin galoofisyada la tuuri karo, waji-xidhka, iwm. inta lagu jiro qaliinka.

Talo soo jeedin: Tijaabooyinka soo saarista RNA waxaa sida ugu fiican loogu sameeyaa qolal kala duwan oo wax lagu sameeyo RNA miiskana waa la nadiifiyaa ka hor tijaabada.

Xiro galoofyada iyo maaskaro la tuuri karo inta lagu jiro tijaabada si loo yareeyo xaalufka RNA ee ay keento hordhaca RNase.

4. Reagen-yada waxa ku wasakhaysan RNase marka la isticmaalayo.

Talo-soo-jeedin: Ku beddel Xayawaanada cusub ee Wadarta Guud ee Go'doonka RNA ee tijaabooyinka la xidhiidha.

5. Tuubooyinka centrifuge, talooyinka, iwm. ee loo isticmaalo wax-ka-beddelka RNA waxay ku wasakhoobeen RNase.

Talo soo jeedin: Xaqiiji in tuubbooyinka centrifuge, talooyinka, pipettes, iwm. ee loo isticmaalo soo saarista RNA ay dhamaantood yihiin kuwo ka madax banaan RNase.

RNA la safeeyey ee la helay waxay saamaysaa tijaabooyinka hoose

RNA oo lagu nadiifiyo tiirka sifaynta, haddii ions milix ah, waxa ku jira borotiinku aad u weyn yahay waxay saamaynaysaa tijaabada hoose, sida: qoraalka dib u noqoshada, Northern Blot et al.

1. RNA-ga la macmalay waxa uu leeyahay hadhaaga ion cusbo.

Talo soo jeedin: Xaqiiji in mugga saxda ah ee ethanol lagu daray Buffer RW2 oo samee 2 tiir nadiifin ah oo dhaqida xawliga centrifugal ee lagu tilmaamay hawlgalka;Haddii ay jiraan wax hadhaaga ion cusbo, u dhaaf tiirka sifaynta Buffer RW2 5 daqiiqo heerkulka qolka oo samee centrifugation si aad u kordhiso ka saarida wasakhowga milixda.

2. Hadhaaga Ethanol ee RNA-ga la macmalay.

Talo soo jeedin: Xaqiiji in ka dib marka la dhaqo RW2 baffer, samee hawlgalka centrifugation tube madhan xawaaraha centrifugation ee lagu tilmaamay hawlgalka, kordhiyo waqtiga hawlgalka centrifugation tube madhan 2 min haddii ay weli jiraan haraaga ethanol, ama ka tago heerkulka qolka 5 daqiiqo ka dib tuubada centrifugation madhan si loo kordhiyo ka saarida ethanolue.

Go'doominta noocyada muunada aashitada nulceic

Xirmooyinka Go'doominta RNA ee Foregene waxay heli karaan wadarta go'doominta / ka saarista RNA ee soo socota maraqa muunada ee soo socda: Unugyada xayawaanka, dhirta, unugyada, fayraska, dhiigga, iwm.

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