Isethi Yekhithi Yokuzihlukanisa Yeseli Ye-RNA Ingqikithi Yekhithi Yokuhlanzwa Yokuzihlukanisa ye-RNA kuseli
Incazelo yekhithi:
I-RNA ehlanzwe kakhulu futhi yekhwalithi ephezulu ingatholakala kumaseli ahlukahlukene akhuliswe ngemizuzu eyi-11.
I-RNase-Free
Isebenza ekamelweni lokushisa (15-25 ℃)
Ngekholomu Yokuhlanza I-DNA
Inani eliphakeme kakhulu lama-RNA
Ukushesha:Qeda ukukhipha emizuzwini eyi-11
Ukuphepha:Ayikho i-Organic chemical
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.
TIkholomu ye-RNA kuphela ingabopha i-RNA ngokuphumelelayo ngefomula eyingqayizivele.Inani elikhulu lamasampula lingacutshungulwa kanyekanye.
Izingxenye zekhithi
| Ukwakheka kwekhithi | RE-03111 | RE-03114 |
| 50 T | 200 T | |
| I-Buffer cRL1* | 25 ml | 100 ml |
| Gcina i-cRL2 | 15 ml | 60 ml |
| Isilondolozi RW1* | 25 ml | 100 ml |
| Isilondolozi RW2 | 24 ml | 96 ml |
| RNase-Free ddH2O | 10 ml | 40 ml |
| Ikholomu Ye-RNA Kuphela | 50 | 200 |
| Ikholomu Yokuhlanza I-DNA | 50 | 200 |
| Isiyalezo | 1 | 1 |
*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).
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.
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.
