Akụkụ ndọta/sel sel/ngụkọta ngwa RNA
Atụmatụ
■ Ọ dị mfe yana ngwa ngwa: enwere ike nweta mkpokọta RNA n'ime nkeji 60.
Nrụpụta dị elu: Ọ nwere ike mezuo ihe achọrọ na ntinye akwụkwọ ntuziaka yana mwepụta ogbe na nyiwe dị iche iche dị elu.
■ Nchekwa na anaghị egbu egbu: Enweghị reagent dị ka phenol/chloroform dị mkpa.
Nkọwapụta
Ụdị: Mpempe bekee ụdị mwepụ.
Sample: anụ ahụ, sel na ọbara.
Ebumnuche: Mkpokọta RNA
Malite olu: 5-20 mg, gafere 1 × 107, 0.1-1.5 ml
Oge ọrụ: 60 min
Ngwa dị n'okpuru ala: RT-PCR/RT-qPCR, owuwu ụlọ akwụkwọ NGS, wdg.
Enwere ike ịhazi ngwaahịa niile maka ODM/OEM. Maka nkọwa,biko pịa Ọrụ ahaziri (ODM/OEM)
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Ewepụtara mkpokọta RNA site na imeju oke 20 mg na TIANGEN Tissue Tissue/Cell/Blood Total RNA Kit na ngwaahịa ndị dị mkpa sitere n'aka ndị na -eweta ngwaahịa ndị ọzọ. Ebufere 5 μl nke 200 μl eluate na 1% electrophoresis gel agarose, 6 V/cm. Mmechi: Nwepụta mmịpụta, ịdị ọcha na nkwụsi ike nke TANGEN Tissue Tissue/Cell/Blood Total RNA Kit dị mma karịa nke ndị na -eweta ngwaahịa ndị ọzọ. |
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Enwetara mkpokọta RNA site na 20 mg nke akụrụ oke na TIANGEN Magnetic Tissue/Cell/Blood Total RNA Kit na TIANGEN TGuide S32 ma ọ bụ Thermo Kingfisher Flex96 n'otu n'otu. Ejiri 5 μl nke 200 μl eluate gaa na 1% agarose gel electrophoresis, 6 V/cm. Ihe akara: TIANGEN D15000 DNA Marker. Mmechi: Magnetik Tissue/Cell/Blood Total RNA Kit nwere ezigbo mmịpụta mkpụrụ na ịdị ọcha na ndị nrụpụta akpaaka dị iche iche. |
A-1 Cell lysis ma ọ bụ homogenization ezughị
---- Belata ojiji nlele, mụbaa ọnụọgụ ihe nchekwa lysis, mụbaa homogenization na oge lysis.
A-2 Ego nlele buru oke ibu
---- Belata ọnụ ọgụgụ nlele ejiri ma ọ bụ mụbaa ọnụọgụ ihe nchekwa lysis.
A-1 lysis sel zuru oke ma ọ bụ homogenization
---- Belata ojiji nlele, mụbaa ọnụọgụ ihe nchekwa lysis, mụbaa homogenization na oge lysis.
A-2 Ego nlele buru oke ibu
---- Biko rụtụ aka na ikike nhazi kacha.
Apụghị A-3 RNA kpamkpam na kọlụm
---- Mgbe agbakwunyere mmiri RNase-Free, hapụ ya maka nkeji ole na ole tupu ịmịcha.
A-4 Ethanol dị na elu
---- Mgbe ịsachasịrị, centrifuge ọzọ wee wepụ ihe ịsa ahụ dịka o kwere mee.
A-5 ewepụghị usoro ọdịbendị kpamkpam
---- Mgbe ị na-anakọta mkpụrụ ndụ, biko gbaa mbọ hụ na ị wepụrụ usoro ọdịbendị ka enwere ike.
A-6 Mkpụrụ ndụ echekwara na RNAstore anaghị agbasasị nke ọma
---- njupụta ụlọ ahịa RNA karịrị nkezi ọdịbendị sel; yabụ ekwesịrị ịbawanye ike centrifugal. A na -atụ aro ka centrifuge na 3000x g.
A-7 Ọdịnaya RNA dị ala na ụba na nlele
---- Jiri nlele dị mma chọpụta ma ọ bụrụ na nlele ahụ kpatara obere mkpụrụ.
A-1 Ihe a abụghị nke ọhụrụ
---- Ekwesịrị idobe anụ ahụ ọhụrụ na nitrogen mmiri mmiri ozugbo ma ọ bụ tinye ya ozugbo na reagent RNAstore iji hụ na mmịpụta ahụ.
A-2 Ego nlele buru oke ibu
---- Belata ego nlele.
A-3 RNase mmetọn
---- Ọ bụ ezie na ihe nchekwa enyere na ngwa ahụ enweghị RNase, ọ dị mfe imetọ RNase n'oge usoro mwepụ yana ekwesịrị iji ya mee nke ọma.
A-4 mmetọ Electrophoresis
---- Dochie ihe nchekwa electrophoresis wee gbaa mbọ hụ na ihe ndị na-eri ihe na ihe nchekwa na-enweghị mmetọ RNase.
A-5 Oke oke maka electrophoresis
---- Belata ego nlele nlele, itinye nke ọma ọ bụla ekwesịghị ịfe 2 μg.
A-1 Ego nlele buru oke ibu
---- Belata ego nlele.
A-2 Ụfọdụ nlele nwere ọdịnaya DNA dị elu ma enwere ike iji DNase gwọọ ya.
Mee ọgwụgwọ DNase na-enweghị RNase na ntinye RNA enwetara, enwere ike iji RNA mee ihe maka nnwale ndị na-esote mgbe ọgwụgwọ gasịrị, ma ọ bụ ihe mkpocha RNA nwere ike mee ka ọ dị ọcha.
Maka iko iko, sie na 150 Celsius maka awa 4. Maka arịa plastik, mikpuru n'ime 0.5 M NaOH maka nkeji iri, wee jiri mmiri na-enweghị RNase sachaa ya nke ọma wee sterilaiz iji wepụ RNase kpamkpam. Ihe reagents ma ọ bụ azịza ejiri mee nnwale ahụ, ọkachasị mmiri, ga -abụrịrị enweghị RNase. Jiri mmiri na-enweghị RNase maka nkwadebe reagent niile (gbakwunye mmiri na karama iko dị ọcha, gbakwunye DEPC na mkpokọta ikpeazụ nke 0.1% (V/V), maa jijiji n'otu abalị na autoclave).
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