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I-RNA ayikhishiwe noma isivuno se-RNA siphansi

Kuvame ukuba nezinto ezihlukahlukene ezithinta ukusebenza kahle kokululama, njengalezi: okuqukethwe kwesampula yezicubu ze-RNA, indlela yokusebenza, ivolumu ye-elution, njll.

1. Ukugeza kweqhwa noma i-cryogenic (4 °C) i-centrifugation yenziwa ngesikhathi sokusebenza.

Okutuswayo: Sebenzisa ekamelweni lokushisa (15-25 ° C) kuyo yonke inqubo, ungabhavi eqhweni kanye ne-centrifuge emazingeni okushisa aphansi.

2. Ukulondolozwa kwesampula okungalungile noma isikhathi sokugcina isampula eseqile.

Okutuswayo: Gcina amasampula ku--80 °C noma ufrize ku-nitrogen ewuketshezi futhi ugweme ukusetshenziswa okuphindaphindiwe kokuqhwaza;zama ukusebenzisa izicubu ezintsha noma amaseli akhulisiwe ukuze kukhishwe i-RNA.

3. Isampula yesampula enganele.

Okutuswayo: Uma wenza izicubu zibe homogenizing, qinisekisa ukuthi izicubu zihlanganiswe ngendlela eyanele nokuthi amaseli ezicubu ahlukaniswe ngokwanele ukuchaza ukukhululwa kwe-RNA.

4. I-eluent ayengezwanga ngendlela efanele.

Okunconyiwe: Qinisekisa ukuthi i-ddH ye-RNase-Free₂U-O wengezwa ngokudonsela phansi maphakathi nolwelwesi lwekholomu yokuhlanza.

5. Ivolumu elungile ye-ethanol ephelele ayizange yengezwe ku-Buffer RL2 noma ku-Buffer RW2.

Isincomo: Landela imiyalelo, engeza ivolumu efanele ye-ethanol ephelele ku-Buffer RL2 kanye ne-Buffer RW2 bese uxuba kahle ngaphambi kokusebenzisa ikhithi.

6. Umthamo wesampula wezicubu awufanelekile.

Isincomo: Sebenzisa i-10-20 mg yezicubu noma (1-5) × 10⁶amaseli nge-500 μl buffer RL1, njengoba ukusetshenziswa kwezicubu ngokweqile kungaholela ekukhishweni kwe-RNA okuncishisiwe.

7. Ivolumu ye-elution engafanele noma ukungezwani okuphelele.

Okutuswayo: Ivolumu ye-elution yekholomu yokuhlanza ingu-50-200 μl;uma umphumela we-elution ungagculisi, kuyanconywa ukuthi kunwetshwe isikhathi sokubeka izinga lokushisa legumbi ngemva kokwengeza i-ddH eshisiwe ye-RNase-Free₂O, isb imizuzu emi-5-10.

8.Ikholomu yokuhlanza inensalela ye-ethanol ngemva kokugeza kwe-Buffer RW2.

Okutuswayo: Uma kunensalela ye-ethanol ngemva kokugeza kwe-Buffer RW2, i-tube centrifugation engenalutho ye-1min, isikhathi sokusebenza kwe-tube centrifugation esingenalutho singanyuswa sibe yi-2min, noma ikholomu yokuhlanza ingabekwa ekamelweni lokushisa imizuzu emi-5 ukuze kukhishwe ngokwanele i-ethanol esele.

I-RNA ehlanziwe yehlisiwe

Ikhwalithi ye-RNA ehlanzekile ihlobene nezici ezifana nokulondolozwa kwesampula, ukungcoliswa kwe-RNase, nokukhohlisa, njll.

1. Amasampula ezicubu awagcinwa ngesikhathi.

Okutuswayo: Uma amasampula ezicubu noma amaseli engasetshenziswa ngesikhathi esifanele ngemva kokuqoqwa, londoloza ngokushesha i-cryopreserve ku- -80 °C noma i-nitrogen ewuketshezi.Ukuze ukhiphe i-RNA, sebenzisa ithishu esanda kuthathwa noma isampula yeseli noma nini lapho kunokwenzeka.

2. Ukuqhwaza okuphindaphindiwe kwamasampula ezicubu.

Okutuswayo: Uma ugcina amasampula ezicubu, kuhle kakhulu ukuwasika abe izingcezu ezincane ukuze alondolozwe, bese ususa ingxenye eyodwa yezingcezu lapho uzisebenzisa ukugwema ukuncibilika okubandayo kwesampula kanye nokonakala kwe-RNA.

3. I-RNase yethulwa noma ingagqoki amagilavu ​​alahlwayo, imaski, njll. phakathi nokuhlinzwa.

Okunconyiwe: Ukuhlolwa kokukhipha i-RNA kwenziwa kangcono kakhulu emagumbini okukhohlisa e-RNA ahlukene futhi ithebula liyasulwa ngaphambi kokuhlolwa.

Gqoka amagilavu ​​alahlwayo kanye nemaski ngesikhathi sokuhlolwa ukuze unciphise ukucekelwa phansi kwe-RNA okubangelwa ukwethulwa kwe-RNase.

4. Ama-reagents angcoliswe yi-RNase ngesikhathi sokusetshenziswa.

Okunconyiwe: Faka esikhundleni Ikhithi Yokuhlukanisa Yezilwane Ephelele Yenani Le-RNA ukuze uthole ukuhlolwa okuhlobene.

5. Amashubhu e-centrifuge, amathiphu, njll. asetshenziswa ekukhohlisweni kwe-RNA angcoliswe yi-RNase.

Okutuswayo: Qinisekisa ukuthi amashubhu e-centrifuge, amathiphu, amapayipi, njll. asetshenziswa ekukhishweni kwe-RNA konke Akuna-RNAse.

I-RNA etholiwe ehlanziwe ithinta ukuhlola okwehla nomfula

I-RNA ehlanjululwe ngekholomu yokuhlanza, uma ama-ion kasawoti, okuqukethwe kwamaprotheni kukukhulu kakhulu kuzothinta isilingo esisezansi nomfula, njengokuthi: ukuloba okuhlanekezelwe,Northern Blot et al.

1. I-RNA ekhishiwe inezinsalela ze-ion kasawoti.

Okunconyiwe: Qinisekisa ukuthi ivolumu elungile ye-ethanol yengezwe ku-Buffer RW2 futhi wenze amakholomu okuhlanza ama-2 ageza ngesivinini esimaphakathi esikhonjiswe ukusebenza;uma kukhona noma iyiphi insalela ye-ion kasawoti, shiya ikholomu yokuhlanza ku-Buffer RW2 imizuzu engu-5 ekamelweni lokushisa futhi wenze i-centrifugation ukwandisa ukukhishwa kokungcoliswa kukasawoti.

2. Izinsalela ze-Ethanol ku-RNA elutioned.

Isincomo: Qinisekisa ukuthi ngemva kokugeza kwe-buffer RW2, yenza i-tube centrifugation operation engenalutho ngesivinini se-centrifugation esiboniswe ukusebenza, ukwandisa isikhathi sokusebenza kwe-tube centrifugation engenalutho kumaminithi angu-2 uma kusekhona insalela ye-ethanol, noma uyishiye ekamelweni lokushisa imizuzu engu-5 ngemva kwe-tube centrifugation engenalutho ukuze ukhulise ukukhishwa kwe-thanol.

Ukuhlukaniswa kwezinhlobo zesampula ze-nulceic acid

I-Foregene's RNA Isolation kits ingathola ukuhlukaniswa/ukukhishwa okuphelele kwe-RNA kulawa masampula alandelayo: Izicubu zezilwane, isitshalo, amaseli, igciwane, igazi, njll.

Ngiyigcina kanjani ikhithi

Ukugcinwa kwezinga lokushisa kwegumbi izinyanga ezingama-24.