Imihlahlandlela yokuhlaziya izinkinga

The following is an analysis of the problems that might be encountered in the extraction of viral RNA. We wish it would be helpful to your experiment. In addition, for other experimental or technical problems other than operating instructions and problem analysis, we have dedicated technical support to help you. Contact us if you need at : 028-83361257or E-mail：Tech@foregene.com。

Ayikho i-RNA engakhishwa noma isivuno se-nucleic acid siphansi

Ngokuvamile kuba nezinto eziningi ezithinta ukusebenza kahle kokululama, njengalezi: isampula yokuqukethwe kwe-RNA, indlela yokusebenza, ivolumu ye-elution, njll..

Ukuhlaziywa kwezimbangela ezivamile:

1.Ibhavu yeqhwa noma izinga lokushisa eliphansi (4 ° C) i-centrifugation ngesikhathi sokusebenza.

Ukusikisela: Izinga lokushisa legumbi (15-25 ° C), ungalokothi ugeze eqhweni kanye nezinga lokushisa eliphansi le-centrifuge.

2. Ukugcinwa kwesampula okungalungile noma isampula yokugcina isikhathi eside kakhulu.

Ukusikisela: Gcina amasampula ku- -80 ° C noma ufrize ku-nitrogen ewuketshezi, futhi ugweme ukusetshenziswa kokuqhwaza okuphindaphindiwe;zama ukusebenzisa amasampula asanda kuqoqwa ukuze kukhishwe i-RNA.

3.Isampula yesampula enganele

Okutuswayo: Sicela uqinisekise ukuthi isampula kanye nesixazululo sokusebenza (i-Linear Acrylamide) kuxutshwe kahle futhi kufakwe ekufukameleni imizuzu eyi-10 ekamelweni lokushisa (15-25 ° C)

4.I-eluent yengezwe ngokungalungile

Okutuswayo: Qiniseka ukuthi i-ddH2O ye-RNase-Free yengezwa phakathi nolwelwesi lwekholomu yokuhlanza.

5.Ivolumu engafanele ye-ethanol engenamanzi ku-Buffer viRW2

Ukusikisela: Sicela ulandele imiyalelo, wengeze umthamo olungile we-ethanol enganamanzi ku-Buffer viRW2 bese uyixuba kahle ngaphambi kokusebenzisa ikhithi.

6.Ukusetshenziswa kwesampula okungalungile.

Isiphakamiso: 200µl wesampula ngo-500μl ngamunye we-Buffer viRL.Ivolumu yesampula eyeqile izoholela ekwehliseni izinga lokukhishwa kwe-RNA.

7.Ivolumu ye-elution engafanele noma ukungezwani okuphelele.

Isiphakamiso: Umthamo ongacacile wekholomu yokuhlanza ngu-30-50μl;uma umphumela we-elution ungagculisi, kuyanconywa ukuthi wengeze i-ddH eshisiwe ngaphambilini ye-RNase-Free₂O futhi wandise isikhathi obeka ekamelweni lokushisa, ezifana 5-10min

8.Ikholomu yokuhlanza inezinsalela ze-ethanol ngemva kokuyihlanza ku-Buffer viRW2.

Isiphakamiso: Uma i-ethanol isasele ngemva kokugeza ku-Buffer viRW2 kanye ne-empty-tube centrifugation imizuzu engu-2, ikholomu yokuhlanza ingashiywa ekamelweni lokushisa imizuzu emi-5 ngemva kokuhlanganisa ishubhu engenalutho ukuze ikhiphe ngokuphelele i-ethanol esele.

Ukuwohloka kwama-molecule e-RNA ahlanzekile

Ikhwalithi ye-RNA ehlanziwe ihlobene nezici ezifana nokugcinwa kwesampula, ukungcoliswa kwe-RNase, nokusebenza.

Ukuhlaziywa kwezimbangela ezivamile:

1.Amasampuli aqoqiwe awalondolozwanga ngesikhathi.

Isiphakamiso: Uma isampula lingasetshenziswa ngesikhathi ngemuva kokuqoqwa, sicela uyigcine ku- -80 ℃ noma i-nitrogen ewuketshezi ngokushesha.Ukuze ukhiphe ama-molecule e-RNA, zama ukusebenzisa amasampula asanda kuqoqwa noma nini lapho kungenzeka.

2.Amasampula aqoqiwe abeqhwa futhi ancibilika ngokuphindaphindiwe.

Isiphakamiso: Gwema ukuqhwaza okuphindaphindiwe nokuncibilika (okungaphezu kokukodwa) phakathi nokuqoqwa nokugcinwa kwesampula, ngaphandle kwalokho isivuno se-nucleic acid sizokwehla.

3.I-RNase yethulwa egunjini lokuhlinzela noma kwakungekho amagilavu ​​alahlwayo, imaski, njll.

Isiphakamiso: Ukukhishwa kokuhlolwa kwama-molecule e-RNA kwenziwa kangcono ekamelweni lokusebenza elihlukile le-RNA, futhi ithebula lokuhlola liyahlanzwa ngaphambi kokuhlolwa.Gqoka amagilavu ​​alahlwayo kanye nemaski ngesikhathi sokuhlolwa ukuze ugweme ukucekelwa phansi kwe-RNA okubangelwa ukwethulwa kwe-RNase.

4.I-reagent ingcoliswe yi-RNase ngesikhathi isetshenziswa.

Isiphakamiso: Faka esikhundleni se-Viral RNA Isolation Kit entsha ukuze uthole ukuhlolwa okuhlobene.

5.Ukungcoliswa kwe-RNase kwamashubhu e-centrifuge, amathiphu e-pipette, njll. Isiphakamiso: Qiniseka ukuthi amashubhu e-centrifuge, amathiphu e-pipette, namapayipi konke Akuna-RNAse.

Ama-molecule e-RNA ahlanziwe athinte ukuhlolwa okungaphansi komfula

Ama-molecule e-RNA ahlanzwe ikholomu yokuhlanza azothinta ukuhlolwa okuya phansi komfula uma kukhona ama-ion kasawoti amaningi noma amaprotheni, afana nalokhu: ukuloba okuhlanekezelwe, i-Northern Blot, njll..

1.Kukhona ama-ion kasawoti asele kuma-athomu e-RNA e-luted.

Okutuswayo: Qiniseka ukuthi ivolumu elungile ye-ethanol enganamanzi yengezwe ku-Buffer viRW2, futhi ugeze ikholomu yokuhlanza kabili ngokwejubane elifanele le-centrifugation emiyalweni yokusebenza;Uma kusekhona ama-ion kasawoti asele, ungakwazi ukwengeza i-Buffer viRW2 kukholamu yokuhlanza, futhi uyishiye ekamelweni lokushisa imizuzu emi-5.Bese wenza i-centrifugation ukuze ususe ukungcoliswa kwama-ion kasawoti ngezinga elikhulu kakhulu

2.Kune-ethanol esele kuma-athomu e-RNA akhishwe

Isiphakamiso: uma usuqinisekisa ukuthi amakholomu okuhlanza ahlanjululwe yi-Buffer viRW2, yenza i- empty-tube centrifugation ngokwesivinini se-centrifugal emiyalweni yokusebenza.Uma isekhona i-ethanol esele, ingashiywa imizuzu emi-5 ekamelweni lokushisa ngemva kwe-centrifugation yeshubhu engenalutho ukuze kukhishwe i-ethanol esele ngezinga elikhulu kakhulu.

Amamanuwali wemiyalo:

I-Viral RNA Isolation Kit Manual