FAQ makaNgwakọta RNA dị iche iche anụmanụ

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A naghị ewepụta RNA ma ọ bụ RNA dị ala

A na-enwekarị ihe dị iche iche na-emetụta arụmọrụ mgbake, dịka: ọdịnaya RNA anụ ahụ, usoro ọrụ, elution volume, wdg.

1. Ice bath ma ọ bụ cryogenic (4 Celsius C) centrifugation mere n'oge ọrụ.

Nkwanye: Na-arụ ọrụ na ụlọ okpomọkụ (15-25 Celsius C) n'ime usoro dum, emela ice bat na centrifuge na obere okpomọkụ.

2. Nchekwa nlele na-ezighi ezi ma ọ bụ oge nchekwa oke.

Nkwanye: Chekwaa ihe nlele na -80 Celsius ma ọ bụ ifriizi na mmiri mmiri nitrogen ma zere iji friza eme ihe ugboro ugboro;gbalịa iji anụ ahụ ọhụrụ ma ọ bụ mkpụrụ ndụ emebere maka mmịpụta RNA.

3. ezughị ezu sample lysis.

Nkwanye: Mgbe ị na-eme ka anụ ahụ dị n'otu, hụ na anụ ahụ na-emekọ ihe nke ọma yana na mkpụrụ ndụ anụ ahụ kewara nke ọma iji kọwaa ntọhapụ nke RNA.

4. A naghị atụkwasị eluent nke ọma.

Nkwanye: Kwenye na ddH enweghị RNase₂A na-agbakwunye O n'ime ala n'etiti akpụkpọ ahụ kọlụm nke ịdị ọcha.

5. Agbakwunyeghị olu ziri ezi nke ethanol zuru oke na Buffer RL2 ma ọ bụ Buffer RW2.

Nkwanye: Soro ntuziaka a, tinye nha ziri ezi nke ethanol zuru oke na Buffer RL2 na Buffer RW2 wee gwakọta nke ọma tupu i jiri ngwa ahụ.

6. Usoro ihe nlele anụ ahụ adịghị mma.

Nkwanye: Jiri 10-20 mg nke anụ ahụ ma ọ bụ (1-5) × 10⁶mkpụrụ ndụ kwa 500 μl ihe nchekwa RL1, n'ihi na iji anụ ahụ gabigara ókè nwere ike ime ka mmịpụta RNA belata.

7. Olu elution na-ezighi ezi ma ọ bụ elution ezughị ezu.

Nkwanye: Oke elution nke kọlụm nchacha bụ 50-200 μl;Ọ bụrụ na mmetụta elution adịghị afọ ojuju, a na-atụ aro ịgbatị oge ntinye ụlọ okpomọkụ mgbe agbakwunyere ddH na-enweghị ọkụ RNase.₂O, ọmụmaatụ maka nkeji 5-10.

8.The kọlụm dị ọcha nwere ethanol fọdụrụnụ mgbe Buffer RW2 saa.

Nkwanye: Ọ bụrụ na e nwere ihe fọdụrụ na ethanol mgbe ịsachara Buffer RW2, centrifugation tube efu maka 1min, enwere ike ịbawanye oge ọrụ centrifugation tube efu ruo 2min, ma ọ bụ enwere ike idobe kọlụm dị ọcha na ụlọ okpomọkụ maka 5 min iji wepụ ethanol fọdụrụnụ nke ọma.

RNA dị ọcha na-eweda ya ala

Àgwà nke RNA a sachara ejikọta na ihe ndị dị ka ichekwa ihe nlele, mmetọ RNase, na nhazi, wdg.

1. A naghị edebe ihe nlele anụ ahụ n'oge.

Nkwanye: Ọ bụrụ na ejighị ihe nlele anụ ahụ ma ọ bụ sel mee ihe n'oge kwesịrị ekwesị mgbe nchịkọtachara, ozugbo cryopreserve na -80 Celsius C ma ọ bụ nitrogen mmiri mmiri.Iji wepụ RNA, jiri anụ ahụ ma ọ bụ ihe nlele cell ewepụtara ọhụrụ mgbe ọ bụla enwere ike.

2. Ugboro ugboro ifriizi-thawing nke anụ ahụ sample.

Nkwanye: Mgbe ị na-echekwa ihe nlele anụ ahụ, ọ kacha mma ịkpụ ya n'ime obere iberibe maka nchekwa, ma wepụ otu n'ime iberibe ahụ mgbe ị na-eji ha ka ịzenarị oyiri ugboro ugboro nke ihe nlele ahụ na mmebi nke RNA.

3. A na-ewebata RNase ma ọ bụ ghara iyi uwe mkpuchi, ihe mkpuchi, wdg n'oge ọrụ ahụ.

Nkwanye: A na-eme nnwale mmịpụta RNA kacha mma n'ime ụlọ njikwa RNA dị iche iche wee kpochapụ tebụl tupu nnwale ahụ.

Yiri gloves na masks enwere ike iwepụ n'oge nnwale ahụ iji belata mmebi RNA kpatara site na iwebata RNase.

4. A na-eji RNase metọọ reagents n'oge eji.

Nkwanye: Dochie jiri ngwa Mkpokọta anụ ọhịa RNA ọhụrụ maka nnwale ndị metụtara ya.

5. Ọkpụkpọ centrifuge, ndụmọdụ, wdg eji eme ihe na RNA na-emetọ ya na RNase.

Nkwanye: Kwenye na tubes centrifuge, ndụmọdụ, pipettes, wdg. ejiri na mmịpụta RNA bụ RNase niile.

RNA emepụtara dị ọcha na-emetụta nnwale ndị dị n'okpuru ala

RNA sachara site na kọlụm dị ọcha, ọ bụrụ na ion nnu, ọdịnaya protein buru ibu ga-emetụta nnwale nke ala, dị ka: ntụgharị ntụgharị, Northern Blot et al.

1. RNA nke elutioned nwere residues ion nnu.

Nkwanye: Kwenye na agbakwunyere oke ethanol ziri ezi na Buffer RW2 ma mee 2 kọlụm nchacha ọcha na ọsọ centrifugal nke egosiri maka ịrụ ọrụ;Ọ bụrụ na enwere ihe fọdụrụ ion nnu, hapụ kọlụm nke ịdị ọcha na Buffer RW2 maka 5 min na ụlọ okpomọkụ ma mee centrifugation iji bulie mwepụ nke mmetọ nnu.

2. Ethanol fọdụrụnụ na elutioned RNA.

Nkwanye: Kwenye na mgbe ịsachara RW2 na-enweghị ihe ọ bụla, rụọ ọrụ centrifugation tube efu na ọsọ centrifugation egosipụtara maka ịrụ ọrụ, mụbaa oge ọrụ centrifugation tube efu ruo 2 min ma ọ bụrụ na ọ ka nwere ihe fọdụrụ na ethanol, ma ọ bụ hapụ ya n'ime ụlọ okpomọkụ maka 5 min mgbe centrifugation efu gachara iji bulie mwepụ nke ethanolue.

Mwepu nke nulceic acid ụdị sample

Ngwa iche iche nke Foregene's RNA nwere ike nweta mkpokọta RNA kewapụ/mwepụta site na isi ihe atụ ndị a: anụ ahụ anụ ahụ, osisi, mkpụrụ ndụ, viral, ọbara, wdg.

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