Escherichia Coli O157: Ngwa Nchọpụta Nucleic Acid H7 (Ụzọ Nyocha Nyocha/Lyophilization PCR Fluorescent)
Nkọwa ngwa:
Nwamba. Mba.FP103
A na-eji ya maka nchọpụta ngwa ngwa na nyocha nke E. coli O157: H7 na nri, nri, mmiri mmiri na ihe atụ gburugburu ebe obibi.
Nkọwa
A na-eji yanchọpụta ngwa ngwa na nyocha nke E. coli O157: H7 na nri, nri nri, mmiri mmiri na ihe atụ gburugburu ebe obibi.
[ụkpụrụ nnwale]
Dị ka ụkpụrụ nke fluorescent PCR technology, kpọmkwem primers na Taqman probes e mere maka kpọmkwem mkpụrụ ndụ ihe nketa nke Escherichia coli O157: H7, na-achọpụtara site a fluorescent PCR ngwá, iji ghọta nchọpụta Escherichia coli O157: H7 Qualitative nchọpụta nke DNA.
Ọdịnaya ngwa
Mara: Agụnyeghị nyocha ọwa ROX.
| Cndị na-akwado | Nkọwapụta | Qịdị n'otu |
| Buffer A | Tube | 1 |
| Ihe nchekwa B | Tube | 1 |
| njikwa dị mma | Tube | 1 |
| Njikwa adịghị mma | Tube | 1 |
Ojiji a tụrụ anya ya
A na-eji ya nchọpụta ngwa ngwa na nyocha nke E. coli O157: H7 na nri, nri nri, mmiri mmiri na ihe atụ gburugburu ebe obibi.
Ọnọdụ Nchekwa na Ụbọchị Mmebi
Chekwaa na -20 ℃ n'ọchịchịrị wee zere ịza oyi na agbaze ugboro ugboro.
Oge nkwado bụ 12ọnwa, na ụbọchị mmepụta gosipụtara na nkwakọ ngwaahịa dị n'elu.
Ngwa na ihe eji eme ihe
Ngwa PCR nke fluorescent, egbe pipette na ndụmọdụ dakọtara, vortex shaker, mini centrifuge.
Ojiji
1. Nhazi ihe nlele
Ụdị ihe atụ 1.1: Ngwa a dabara adaba maka nri, nri, mmiri mmiri na ihe ndị ọzọ a na-enyo enyo na Escherichia coli O157: H7 na-emerụ ya.Maka ngwaahịa anụ a na-eme nke ọma, ihe ọṅụṅụ na ihe ndị ọzọ nwere pigmenti, ọ dị mkpa ka a kpochaa ha ka ọ ghara imetụta nchịkọta mgbaàmà fluorescence.
1.2 Nhazi nlele: zoo aka na "GB 4789.10-2016 Food Safety National Standard Food Microbiological Examination of Escherichia coli O157: H7 Test" maka nkwadebe nlele, omenala ịba ụba na ikewapụ Escherichia coli O157: H7.
- Nucleic acid mmịpụta
Were 20 ml nke ihe oriri na-edozi ahụ na tube centrifuge 1.5 ml, tinye 200 μL nke microbial lysate (a na-achọ ngwa mgbakwunye), vortex maka 30 sekọnd, centrifuge nkenke, ma wepụ ya.
Nkwupụta: Mwepụta nke nucleic acid na lysate kwesịrị imecha n'ime nkeji 10, enweghịkwa ike ịchekwa ya ogologo oge.
3. Nucleic Acid Amplification
3.1 Gbanwuo ngwa PCR fluorescent quantitative maka ojiji.
Buffer A na Buffer B sitere na ngwa ahụ, gbazee ha nke ọma, ma kpachie obere oge.Tinye 18 μL Buffer A na 2 μL Buffer B na tube mmeghachi omume PCR ọ bụla.Mgbe ahụ tinye 5 ml nke ọ bụla n'ime njikwa na-adịghị mma, nucleic acid amịpụtara, na njikwa dị mma n'ime tubes mmeghachi omume PCR, kpuchie tubes, na centrifuge obere oge.
3.3 Nyefee PCR mmeghachi omume tube na a fluorescent PCR igwe, na-eji usoro ndị a na-esonụ amplification nnwale: họrọ 25 mL maka mmeghachi omume usoro, na-anakọta fluorescence mgbaàmà na 60 Celsius C maka onye ọ bụla okirikiri, na họrọ FAM maka nchọpụta ọwa.
| Nzọụkwụ | Mmemme | Ọnụọgụ okirikiri |
| 1 | 37 ℃ 5 nkeji | 1 |
| 2 | 9 5 ℃ 3 nkeji | 1 |
| 3 | 95°C 15s | 40 |
| 60℃ 30s (na-anakọta fluorescence) |
Ntuziaka maka nyocha nsogbu
The following is an analysis of the problems that might be encountered in the extraction of viral RNA. We wish it would be helpful to your experiment. In addition, for other experimental or technical problems other than operating instructions and problem analysis, we have dedicated technical support to help you. Contact us if you need at : 028-83361257or E-mail：Tech@foregene.com。
Enweghị RNA enweghị ike ịwepụta ma ọ bụ mkpụrụ nke nucleic acid dị ala
A na-enwekarị ọtụtụ ihe na-emetụta arụmọrụ mgbake, dị ka: sample ọdịnaya RNA, usoro ọrụ, elution volume, wdg.
Nyocha nke ihe na-akpatakarị:
1.Ice bath ma ọ bụ obere okpomọkụ (4 Celsius C) centrifugation n'oge ọrụ.
Aro: Arụ ọrụ ụlọ okpomọkụ (15-25 Celsius C), ọ dịghị mgbe ịsa ahụ na ice na obere okpomọkụ centrifuge.
2. Nchekwa ihe nlere na-ezighi ezi ma ọ bụ nchekwa nchekwa maka ogologo oge.
Aro: Chekwaa nlele na -80 Celsius ma ọ bụ ifriizi na mmiri mmiri nitrogen, ma zere iji friza eme ihe ugboro ugboro;nwaa iji ihe nlele anakọtara ọhụrụ maka mmịpụta RNA.
3.Ezughị ezu sample lysis
Nkwanye: Biko hụ na agwakọtala ihe nlele na ihe ngwọta na-arụ ọrụ (Linear Acrylamide) ma tinye ya maka nkeji 10 n'ime ụlọ okpomọkụ (15-25 Celsius C).
4.Agbakwunyere eluent na-ezighi ezi
Nkwanye: Gbaa mbọ hụ na agbakwunyere RNase-Free ddH2O n'etiti akpụkpọ ahụ nke kọlụm nchacha.
5.Ezigbo olu nke anhydrous ethanol na Buffer viRW2
Aro: Biko soro ntuziaka a, tinye nha kwesịrị ekwesị nke ethanol anhydrous na Buffer viRW2 wee gwakọta ha nke ọma tupu i jiri ngwa ahụ.
6.Ezigbo sample ojiji.
Aro: 200µl nke nlele kwa 500μl nke ihe nchekwa viRL.Oke sample oke ga-ebute mbelata mmịpụta RNA.
7.Ezigbo elution olu ma ọ bụ ezughị ezu elution.
Aro: Oke eluent nke kọlụm nchacha bụ 30-50μl;Ọ bụrụ na mmetụta elution adịghị afọ ojuju, a na-atụ aro ka ịtinye ddH na-enweghị ọkụ ọkụ nke RNase.2O ma gbatịa oge idowe na ụlọ okpomọkụ, dị ka 5-10min
8.Purification kọlụm nwere ethanol residue mgbe rinsing na Buffer viRW2.
Aro: Ọ bụrụ na ethanol ka dịgidere ka emechara ya na Buffer viRW2 na centrifugation tube efu maka nkeji 2, enwere ike ịhapụ kọlụm dị ọcha na ụlọ okpomọkụ maka 5min ka centrifugation na-enweghị tube ga-ewepụ kpamkpam ethanol fọdụrụnụ.
Mmebi nke ụmụ irighiri ihe RNA dị ọcha
Ọdịmma RNA emechara dị ọcha metụtara ihe ndị dị ka nchekwa nlele, mmetọ RNase, na ọrụ.
Nyocha nke ihe na-akpatakarị:
1. A naghị echekwa ihe ndị a na-anakọta n'oge.
Aro: Ọ bụrụ na ejighị ihe nlele ahụ n'oge nchịkọta, biko chekwaa ya na -80 ℃ ma ọ bụ nitrogen mmiri mmiri ozugbo.Maka mmịpụta nke ụmụ irighiri ihe RNA, gbalịa iji ihe nlele anakọtara ọhụrụ mgbe ọ bụla enwere ike.
2.Achịkọtara samples bụ kefriza na thawing ugboro ugboro.
Aro: Zenarị oyi na agbaze ugboro ugboro (ọ dịghị ihe karịrị otu ugboro) n'oge nchịkọta na nchekwa, ma ọ bụghị ya, mkpụrụ nke nucleic acid ga-ebelata.
E webatara 3.RNase n'ime ụlọ ịsa ahụ ma ọ bụ enweghị uwe, ihe mkpuchi, wdg.
Aro: Mwepụta nke nnwale ụmụ irighiri ihe RNA kacha mma n'ime ụlọ ọrụ RNA dị iche, a na-ehichakwa tebụl nnwale tupu nnwale ahụ.Yiri gloves na masks enwere ike iwepụ n'oge nnwale ahụ ka ịzenarị mmebi RNA nke iwebata RNase kpatara.
4.The reagent na-emerụ site RNase n'oge ojiji.
Aro: Dochie na Viral RNA Isolation Kit ọhụrụ maka nnwale ndị metụtara ya.
5.The RNase mmetọ nke centrifuge tubes, pipette ndụmọdụ, wdg Aro: Jide n'aka na centrifuge tubes, pipette ndụmọdụ, na pipettes niile bụ RNase-free.
Ụmụ irighiri ihe RNA emechara emetụta nnwale ndị dị n'okpuru ala
Ngwakọta RNA ndị a sachara site na kọlụm dị ọcha ga-emetụta nnwale ndị dị n'okpuru ma ọ bụrụ na enwere ion nnu ma ọ bụ protein dị ukwuu, dị ka: ntụgharị ntụgharị, Northern Blot, wdg.
1.There fọdụrụ ion nnu na ụmụ irighiri RNA eluted.
Nkwanye: Gbaa mbọ hụ na agbakwunyere oke nke ethanol anhydrous na Buffer viRW2, wee sachaa kọlụm nke ịdị ọcha ugboro abụọ dịka ọsọ centrifugation ziri ezi na ntuziaka ọrụ; Ọ bụrụ na ion nnu ka fọdụrụ, ị nwere ike itinye Buffer viRW2 na kọlụm nchacha, wee hapụ ya na ụlọ okpomọkụ maka 5min.Mgbe ahụ mee centrifugation iji wepụ mmetọ ion nnu ruo n'ókè kasị ukwuu
2.There fọdụrụ ethanol na eluted RNA ụmụ irighiri
Aro: ozugbo na-akwado na Buffer viRW2 sachara kọlụm nchacha, mee centrifugation tube efu dịka ọsọ centrifugal na ntuziaka ọrụ.Ọ bụrụ na ethanol ka fọdụrụ, enwere ike ịhapụ ya maka nkeji 5 n'ime ụlọ okpomọkụ mgbe centrifugation na-adịghị na tube ga-ewepụ ethanol fọdụrụnụ ruo n'ókè kasị ukwuu.
Akwụkwọ ntuziaka:
Akwụkwọ ntuziaka ngwa mwepu RNA dịpụrụ adịpụ
