Cadka Magnetic/Unug/Dhiig Wadarta RNA
Astaamaha
■ Fudud oo degdeg ah: Wadarta guud ee RNA waxaa lagu heli karaa 60 daqiiqo gudahood.
Through Wax-soo-saar sare: Waxay buuxin kartaa shuruudaha soo-saaridda gacanta iyo sidoo kale soo-saaridda dufcadaha kala duwan ee aaladaha kala-soocidda badan.
■ Ammaan ah oo aan sun ahayn: Looma baahna reagent sida phenol/chloroform.
Faahfaahinta
Nooca: Soosaarka nooca kuul magnetic.
Muunad: Cadka, unugyada iyo dhiigga.
Bartilmaameed: Wadarta RNA
Mugga bilowga: 5-20 mg, ha dhaafin 1 × 107, 0.1-1.5 ml
Waqtiga hawlgalka: 60 min
Codsiyada hoose: RT-PCR/RT-qPCR, dhismaha maktabadda NGS, iwm.
Dhammaan alaabooyinka waxaa loo habeyn karaa ODM/OEM. Faahfaahinta,fadlan guji Adeegga La -habeeyey (ODM/OEM)
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Wadarta RNA waxaa laga soo saaray 20 mg beerka jiirka oo ay ku jirto TIANGEN Cadka Magnetic Cell/Cell/Blood Total RNA Kit iyo alaabada ku habboon alaab -qeybiyeyaasha kale. 5 μl oo ah 200 μl eluate waxaa lagu raray 1% agrose gel electrophoresis, 6 V/cm. Gunaanad: Soo -saaridda soo -saaridda, daahirnimada iyo deggenaanshaha TIANGEN Cadka magnetka/Unugga/Dhiigga Wadarta RNA ayaa ka fiican kuwa alaab -qeybiyeyaasha kale. |
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Wadarta RNA waxaa laga soo saaray 20 mg oo kelyo jiir ah oo wata TIANGEN Cadka magnetic/Cell/Blood Total RNA Kit ee TIANGEN TGuide S32 ama Thermo Kingfisher Flex96 siday u kala horreeyaan. 5 μl oo ah 200 μl eluate waxaa lagu raray 1% agrose gel electrophoresis, 6 V/cm. Sumade: TIANGEN D15000 DNA Sumade. Gunaanad: Cadka Magnetka/Unugga/Dhiigga Wadarta RNA Kit wuxuu leeyahay soosaarid soo saarid wanaagsan iyo daahirsanaanta soosaarayaasha otomaatiga ah ee kala duwan. |
A-1 Lysis cell ama homogenization kuma filna
---- Yaree isticmaalka muunadda, kordhi xaddiga kaydinta lysis, kordhiso isugeynta iyo waqtiga lysis.
A-2 Tirada muunaddu aad bay u badan tahay
---- Yaree cadadka muunada la isticmaalay ama la kordhiyo tirada kaydinta lysis.
A-1 Lysis unug oo aan ku filnayn ama isugeyn
---- Yaree isticmaalka muunadda, kordhi xaddiga kaydinta lysis, kordhiso isugeynta iyo waqtiga lysis.
A-2 Tirada muunaddu aad bay u badan tahay
---- Fadlan tixraac awoodda wax-qabadka ugu badan.
A-3 RNA gebi ahaanba lagama soo qaadin tiirka
---- Kadib markaad ku darto biyaha RNase-Free, ka tag dhowr daqiiqo ka hor intaadan ku shubin.
A-4 Ethanol ee ugu sarreeya
---- Ka dib markii aad biyo raacisay, mar kale centrifuge ka saar bakhaarka dhaqidda intii suurtogal ah.
A-5 Dhaqanka dhexdhexaadka ah gebi ahaanba lagama saaro
---- Markaad uruurinayso unugyo, fadlan hubso inaad ka saarto dhexdhexaadinta dhaqanka intii suurtogal ah.
A-6 Unugyada lagu kaydiyo RNAstore si fiican looma darin
---- Cufnaanta RNAstore-ku waxay ka weyn tahay celceliska dhaqanka unugyada; sidaas darteed waa in la kordhiyaa ciidanka dhexe. Waxaa lagu talinayaa in centrifuge at 3000x g.
A-7 Wax yar oo RNA ka kooban iyo tiro badan oo muunadda ah
---- Isticmaal muunad togan si aad u go'aamiso in dhalidda hooseysa ay keentay sambalku.
A-1 Maaddadu ma aha mid cusub
---- Unugyada cusub waa in lagu kaydiyo nitrogen dareere ah isla markiiba ama isla markiiba lagu rido reagent RNAstore si loo hubiyo saamaynta soo saaridda.
A-2 Tirada muunaddu aad bay u badan tahay
---- Yaree cadadka saamiga.
A-3 RNase wasakhaysann
---- Inkastoo kaydka ku jira xirmada uusan ku jirin RNase, way fududahay in la sumoobo RNase inta lagu jiro habka soo saaridda waana in si taxaddar leh loo maareeyaa.
A-4 Nadiifinta Electrophoresis
---- Beddel keydka korontada oo hubso in alaabta wax-cunaysa iyo Kaydinta Loading ay ka madax bannaan yihiin faddaraynta RNase.
A-5 Rarid aad u badan oo loogu talagalay electrophoresis
---- Hoos u dhig xaddiga rarista muunadda, ceel kasta rarantiisu waa inaysan dhaafin 2 μg.
A-1 Tirada muunaddu aad bay u badan tahay
---- Yaree cadadka saamiga.
B
-U samee daaweynta DNase-ka-dhaafka ah ee DNase xalka RNA ee la helay, RNA-na waxaa si toos ah loogu adeegsan karaa tijaabooyinka xiga daaweynta kadib, ama waxaa lagu sii nadiifin karaa xirmooyinka is-nadiifinta RNA.
Muraayadaha dhalooyinka, waxaa lagu dubay 150 ° C 4 h. Weelasha balaastigga ah, oo lagu dhex milay 0.5 M NaOH muddo 10 daqiiqo ah, ka dibna si fiican loogu maydhay biyo aan RNase lahayn ka dibna jeermis si looga saaro RNase. Soo -saarayaasha ama xalka loo adeegsaday tijaabada, gaar ahaan biyaha, waa inay ka madax bannaan yihiin RNase. U isticmaal biyo aan RNase lahayn dhammaan diyaarinta reagent (ku dar biyo dhalo galaas nadiif ah, ku dar DEPC xaddiga ugu dambeeya ee 0.1% (V/V), rux habeen iyo autoclave).
Qaybaha alaabta
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