Izinga elihle liza kuqala ngalo;isevisi ihamba phambili;inhlangano ukubambisana” ifilosofi yethu yebhizinisi ehlala iqashelwa futhi ilandelwa inkampani yethu Yomakhiqizi Osebenza Kakhulu wase-China we-Bioer Bsc53 Magabio Plus Total Rna Purification Kit, Sikholelwa ukuthi usizo lwethu olufudumele nolufanelekayo luzokulethela izinto ezimangalisayo ezingalindelekile kanye nenhlanhla.
Izinga elihle liza kuqala ngalo;isevisi ihamba phambili;inhlangano ukubambisana” kuyifilosofi yethu yebhizinisi ehlala ibonwa futhi ilandelwa inkampani yethuI-China Extraction Reagent, Ubuhlalu obuzibuthe, Ngokuhlanganisa ezokukhiqiza nemikhakha yezohwebo zangaphandle, singanikeza izixazululo eziphelele zamakhasimende ngokuqinisekisa ukulethwa kwemikhiqizo efanele nezisombululo endaweni efanele ngesikhathi esifanele, okusekelwa ulwazi lwethu oluningi, amandla okukhiqiza anamandla, ikhwalithi engaguquki, izimpahla ezihlukene kanye nokulawula izitayela zemboni kanye nokuvuthwa kwethu ngaphambi nangemuva kwezinsizakalo zokuthengisa.Sithanda ukwabelana ngemibono yethu nawe futhi siyakwamukela ukuphawula kwakho nemibuzo.

Incazelo

Le khithi isebenzisa ikholomu yokuphotha kanye nefomula eyakhiwe yi-Foregene, engakhipha ngempumelelo i-RNA ephezulu nekhwalithi ephezulu kumaseli akhuliswe ngamapuleti emithombo engu-96, 24, 12, kanye ne-6.

Ikhithi inikeza Ikholomu Yokuhlanza I-DNA esebenza kahle, engahlukanisa kalula i-supernatant ne-cell lysate, ibophe futhi isuse i-genomic DNA.Ukusebenza kulula futhi konga isikhathi.

Ikholomu ye-RNA kuphela ingahlanganisa ngempumelelo i-RNA ngefomula ehlukile.Inani elikhulu lamasampula lingacutshungulwa kanyekanye.

Izingxenye zekhithi

*Sicela ugqoke amagilavu ​​futhi uthathe izinyathelo zokuvikela ngesikhathi sokusebenza njengoba i-Buffer cRL1 ne-Buffer RW1 iqukethe usawoti we-chaotropic ocasulayo.

Izici&izinzuzo

■ Yonke inqubo iqhutshwa ekamelweni lokushisa (15-25 ℃), ngaphandle kokugeza kweqhwa kanye nokushisa okuphansi kwe-centrifugation.
■ Ikhithi yonke ayinayo i-RNase-Free, asikho isidingo sokukhathazeka ngokuwohloka kwe-RNA.
■ Ikholomu Yokuhlanza I-DNA ibopha ngokukhethekile i-DNA, ukuze ikhithi ikwazi ukususa ukungcola kwe-DNA ye-genomic ngaphandle kokwengeza i-DNase eyengeziwe.
■ Isivuno esikhulu se-RNA: Ikholomu ye-RNA kuphela kanye nefomula eyingqayizivele ingahlanza i-RNA ngokuphumelelayo.
■ Isivinini esisheshayo: kulula ukusebenza futhi singaqedwa ngemizuzu eyi-11.
■ Ukuphepha: Asikho i-reagent ephilayo edingekayo.
■ Ikhwalithi ephezulu: I-RNA ehlanziwe imsulwa kakhulu, ayinamaprotheni nokunye ukungcola, futhi ingahlangabezana nokuhlola okuhlukahlukene okulandelayo.

Isicelo sekhithi

Ilungele ukukhishwa nokuhlanzwa kwe-RNA ephelele kumaseli akhulisiwe kumapuleti angama-96, 24, 12, kanye namapuleti angu-6.

Ukugeleza komsebenzi

Umdwebo

Umdwebo webhethri yejeli ye-agarose ye-Cell Total RNA Isolation Kit iphathe izinombolo ezingenhla ezihlukene zamaseli, ukukhishwa kwevolumu engu-20μl, thatha u-2μl isamba esihlanziwe se-RNA 1%.

Isitoreji nempilo yeshelufu

Ikhithi ingagcinwa izinyanga eziyi-12 ekamelweni lokushisa (15-25 ℃) noma 2-8 ℃ isikhathi eside (izinyanga ezingama-24).

I-Buffer cRL1 ingagcinwa ku-4 ℃ inyanga engu-1 ngemva kokwengeza i-2-hydroxy-1-ethanethiol(ongakukhetha).Ikhwalithi enhle iqala;isevisi ihamba phambili;inhlangano ukubambisana” ifilosofi yethu yebhizinisi ehlala iqashelwa futhi ilandelwa inkampani yethu Yomakhiqizi Osebenza Kakhulu wase-China we-Bioer Bsc53 Magabio Plus Total Rna Purification Kit, Sikholelwa ukuthi usizo lwethu olufudumele nolufanelekayo luzokulethela izinto ezimangalisayo ezingalindelekile kanye nenhlanhla.
Ikhwalithi ephezuluI-China Extraction Reagent, Ubuhlalu obuzibuthe, Ngokuhlanganisa ezokukhiqiza nemikhakha yezohwebo zangaphandle, singanikeza izixazululo eziphelele zamakhasimende ngokuqinisekisa ukulethwa kwemikhiqizo efanele nezisombululo endaweni efanele ngesikhathi esifanele, okusekelwa ulwazi lwethu oluningi, amandla okukhiqiza anamandla, ikhwalithi engaguquki, izimpahla ezihlukene kanye nokulawula izitayela zemboni kanye nokuvuthwa kwethu ngaphambi nangemuva kwezinsizakalo zokuthengisa.Sithanda ukwabelana ngemibono yethu nawe futhi siyakwamukela ukuphawula kwakho nemibuzo.

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-RNase-Free ddH₂U-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 ukukhishwa okungaphelele.

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.

Okutuswayo: Qinisekisa ukuthi ngemva kokugeza i-buffer RW2, yenza umsebenzi ongenalutho we-tube centrifugation ngesivinini se-centrifugation esikhonjiswe ukusebenza, wandise isikhathi sokusebenza kwe-tube centrifugation esingenalutho sibe yimizuzu emi-2 uma kusekhona insalela ye-ethanol, noma uyishiye ekamelweni lokushisa amamitha angu-5.