Ngwa ihe mmịpụta DNA/RNA viral
Ngwa a dabara adaba maka mmịpụta ngwa ngwa nke DNA/RNA viral dị ọcha site na nlele dị ka swabs nasopharyngeal, swabs gburugburu ebe obibi, ihe ndị na-ahụ maka ọdịbendị cell, na anụ ahụ na-ahụ maka ihe ndị na-emepụta ihe.Ngwa ahụ dabere na teknụzụ na-asachapụ akpụkpọ anụ silica nke na-ewepụ mkpa ọ dị iji phenol/chloroform organic solvents ma ọ bụ mmiri ozuzo mmanya na-ewe oge iji wepụta nje virus DNA/RNA dị elu.Acid nucleic enwetara enweghị adịghị ọcha ma dị njikere maka ojiji na nnwale mgbada dị ka ntụgharị ntụgharị, PCR, RT-PCR, PCR ezigbo oge, usoro ọgbọ na-abịa (NGS), na Northern blot.
Ọnọdụ nchekwa
Chekwaa na 15 ~ 25 ℃, ma bufee ya na ụlọ okpomọkụ
Ngwa
| Ngwa | 100RXNS |
| Ihe nchekwa VL | 50 ml |
| Ihe nchekwa RW | 120 ml |
| ddH2 O na-enweghị RNase | 6 ml |
| Ogidi RNA FastPure | 100 |
| tubes mkpokọta (2ml) | 100 |
| Tubes mkpokọta enweghị RNase (1 .5ml) | 100 |
Ihe nchekwa VL:Nye ebe obibi maka lysis na njide.
Ihe nchekwa RW:Wepu protein fọdụrụnụ na adịghị ọcha ndị ọzọ.
ddH2O na-enweghị RNase:Elute DNA/RNA site na akpụkpọ ahụ dị na kọlụm ọkpụkpụ.
Ogidi RNA FastPure:Kpọmkwem adsorb DNA/RNA.
Tubes mkpokọta 2 ml:Anakọta filtrate.
Tubes mkpokọta enweghị RNase 1.5 ml:Anakọta DNA/RNA.
Ngwa
Nasopharyngeal swabs, gburugburu ebe obibi swabs, cell omenala supernatants, na anụ ahụ homogenate supernatants.
Mater jikere onwe yaala
Ndụmọdụ pipette na-enweghị RNase, 1.5 ml tubes sentrifuge na-enweghị RNase, centrifuge, igwekota vortex, na pipettes.
Usoro nnwale
Mee usoro ndị a niile na kabinet biosafety.
1. Tinye 200 μl nke ihe nlele ahụ na tube centrifuge na-enweghị RNase (mebere PBS ma ọ bụ 0.9% NaCl ma ọ bụrụ na enweghi ihe atụ zuru oke), tinye 500 μl nke Buffer VL, jikọta nke ọma site na vortexing maka 15 - 30 sec, na centrifuge. nkenke iji nakọta ngwakọta na ala nke tube.
2. Tinye FastPure RNA kọlụm na mkpokọta Tubes 2 ml.Nyefee ngwakọta site na Nzọụkwụ 1 gaa na FastPure RNA Columns, centrifuge na 12,000 rpm (13,400 × g) maka 1 min, ma tụfuo nzacha.
3. Tinye 600 μl nke Buffer RW na FastPure RNA Columns, centrifuge na 12,000 rpm (13,400 × g) maka 30 sec, ma tụfuo nzacha.
4. Tinyegharịa nzọụkwụ 3.
5. Centrifuge kọlụm efu na 12,000 rpm (13,400 × g) maka 2 min.
6. Jiri nlezianya nyefee kọlụm FastPure RNA n'ime Tubes mkpokọta na-enweghị RNase ọhụrụ 1.5 ml (nke a na-enye na kit), wee tinye 30 - 50 μl nke ddH2O na-enweghị RNase n'etiti akpụkpọ ahụ na-emetụghị kọlụm ahụ.Kwe ka iguzo na ụlọ okpomọkụ maka 1 min na centrifuge na 12,000 rpm (13,400 × g) maka 1 min.
7. Tụfuo kọlụm FastPure RNA.Enwere ike iji DNA/RNA ozugbo maka nyocha ndị na-esote, ma ọ bụ chekwaa na -30 ~ -15°C maka obere oge ma ọ bụ -85 ~-65°C maka ogologo oge.
Ihe ndetu
Maka iji naanị nyocha.Ọ bụghị maka ojiji na usoro nyocha.
1. Hazie samples ka ụlọ okpomọkụ tupu.
2. Nje na-efe efe nke ukwuu.Biko hụ na ewerela nlezianya nchekwa niile dị mkpa tupu nnwale ahụ.
3. Zenarị ntu oyi ugboro ugboro na agbaze nke ihe nlele ahụ, n'ihi na nke a nwere ike ibute mbibi ma ọ bụ belata mkpụrụ nke DNA/RNA viral ewepụtara.
4. Ngwa ejikere onwe ya gụnyere ndụmọdụ pipette na-enweghị RNase, tubes centrifuge na-enweghị RNase 1.5 ml, centrifuge, mixer vortex, na pipettes.
5. Mgbe ị na-eji ngwa ahụ, yiri uwe ụlọ nyocha, uwe mkpuchi latex enwere ike ịtụfu, na ihe mkpuchi nwere ike ịpụpụ ma jiri ihe oriri na-enweghị RNase belata ohere nke mmetọ RNase.
6. Mee usoro niile na ụlọ okpomọkụ ọ gwụla ma akọwapụtara ya.
Mechanism & Usoro ọrụ
