Poly A Umthwali RNA

Inombolo yekati: HC4001A

I-Poly A, i-polyadenylate, ngumxube we-100 ~ 10000 polyadenylates, eyenziwa yi-polynucleotide phosphorylase in vitro.Kwi-vivo, i-poly (a) yongezwa kwi-3-terminal ye-mRNA nge-enzyme ukuphucula ukuzinza kwe-mRNA.Ekusetyenzisweni kwe-nucleic acid extraction, ukongeza i-poly A kwi-lysate okanye isisombululo sokubopha kunokuphucula isivuno se-DNA kunye ne-RNA.Indlela yokusebenzayepoly A iphucula isivuno senucleic acid ngolu hlobo lulandelayo:

1. Uqhagamshelwano olugcweleyo kunye ne-adsorption ephezulu yamanqaku.Uninzi lwamanqaku e-polypropylene anombane omileyo kumphezulu, oya kuthi adsorb i-nucleic acids.Umthwali we-RNA unokuzalisa eziiziphumo ze-adsorption kunye nokunciphisa ilahleko ye-nucleic acid ekujoliswe kuyo.

2. Yenza i-nucleases yokulandelela ingasebenzi: Kukho ii-nuclease ezahlukeneyo kwiisampulu zebhayoloji kunyeokusingqongileyo.I-Poly A inokwenza i-nuclease trace isebenze ekutsalweni okanye kumanyathelo okugcinaukuphucula isivuno kunye nokuzinza kwe-nucleic acid ekujoliswe kuyo.

3. Ukunyibilika: Kwinyathelo lokucocwa kwe-asidi ye-nucleic ye-alcohol mediated precipitation okanye ukubophelela, i-poly A inokukhawulelana ne-nucleic acid ekujoliswe kuyo okanye yenze amasuntswana e-polymer ukuya.ukuphucula ukuchacha.

Indlela yokusetyenziswa

Thatha isixa esifanelekileyo somgubo lyophilized, yongeza DEPC baphathwe amanzi okanye isisombululo ityuwa guanidine ukubayinyibilikise kwi-0.1-1ug/uL, kwaye emva koko uyipakishe kancinci kwaye uyigcine ku -20°C.

Usetyenziso

I-1.Virus DNA / RNA extraction: ukongeza i-1-5ug Carrier RNA kwi-lysate inokuphucula isivuno se-RNA / DNA, izinzile i-nucleic acid ekujoliswe kuyo kwaye igweme ukuthotywa kwe-nucleic acid ecocekileyo ngexesha lokugcinwa.

2. Kwi-micro DNA / RNA extraction by column membrane method (<1ug), ukongeza i-RNA ye-carrier kwi-1-5ug ilungele ukuphucula isivuno se-nucleic acid.

3. Kwi-alcohol mediated nucleic acid precipitation kunye nesinyathelo sokugxininiswa, ukongezwa kwe-1-2ug carrier RNA kunceda ukuphucula ukubuyiswa kwecandelo elifutshane le-RNA.

4. Kwi-probe ye-probe ye-PCR reaction solution, ukongeza i-10-100ng ye-RNA ye-carrier kwi-reaction solution luncedo ekuphuculeni uvakalelo kunye nokunciphisa i-C._Tixabiso.