Ngwa Genotyping òké

Mpempe nwamba: HCR2021A

Ngwaahịa a bụ ngwa emere maka njirimara ngwa ngwa nke genotypes òké, gụnyere mmịpụta crude DNA na sistemụ mmụba PCR.Enwere ike iji ngwaahịa a maka nkwalite PCR ozugbo site na ọdụ òké, ntị, mkpịsị ụkwụ na anụ ahụ ndị ọzọ mgbe ọ gbasasịrị mfe nke Lysis Buffer na Proteinase k.Enweghị mgbari nri abalị, mmịpụta phenol-chloroform ma ọ bụ nchacha kọlụm, nke dị mfe ma na-ebelata oge nnwale.Ngwaahịa a dabara adaba maka mmụba nke iberibe ebumnuche ruru 2kb yana mmeghachi omume PCR multiplex nwere ihe ruru ụzọ abụọ 3.Ngwakọta 2 × Mouse Tissue Direct PCR nwere DNA polymerase emebere site na mkpụrụ ndụ ihe nketa, Mg²⁺, dNTPs na usoro nchekwa ihe kachasị mma iji nye arụmọrụ mmụba dị elu na ntachi obi, ka enwere ike ime mmeghachi omume PCR site na ịgbakwunye template na primers na rehydrating ngwaahịa na 1 ×.Ngwaahịa PCR ejiri ngwaahịa a gbasaa nwere ntọala “A” ama ama na njedebe 3′ ma enwere ike iji ya ozugbo maka cloning TA mgbe emechara ya ọcha.

Ngwa

Ọnọdụ Nchekwa

Ekwesịrị ịchekwa ngwaahịa na -25 ~ -15 ℃ maka afọ 2.Mgbe thawing, Lysis Buffer nwere ike ịchekwa na 2 ~ 8 ℃ maka obere oge otutu ojiji, na mix ọma mgbe eji.

Ngwa

Ngwaahịa a dabara adaba maka nyocha knockout òké, nchọpụta transgenic, genotyping na ndị ọzọ.

Atụmatụ

1.Ọrụ dị mfe: ọ dịghị mkpa wepụ DNA genomic;

2.Ngwa sara mbara: adabara maka mmụba ozugbo nke anụ ahụ òké dị iche iche.

Ntuziaka

1.Ntọhapụ nke genomic DNA

1) Nkwadebe nke lysate

A na-akwado lysate anụ ahụ dị ka ọnụọgụ nke ihe nlele òké ga-edobe (a ga-akwadebe anụ ahụ lysate na saịtị dị ka usoro onunu si dị ma gwakọta ya nke ọma maka ojiji), yana oke reagents chọrọ maka otu nlele bụ ndị a:

2) Nkwadebe ihe atụ na Lysis

Eji anụ ahụ akwadoro

Were ihe nlele anụ ahụ kwesịrị ekwesị na tubes centrifuge dị ọcha, tinye 200μL nke anụ ahụ lysate ọhụrụ na tube centrifuge ọ bụla, vortex na ịma jijiji, wee tinye ya na 55 ℃ maka 30mins, wee kpoo ọkụ na 98 ℃ maka 3mins.

3) Centrifugation

Ghichaa lysate nke ọma na centrifuge na 12,000 rpm maka 5mins.Enwere ike iji supernatant dị ka ndebiri maka nkwalite PCR.Ọ bụrụ na achọrọ template maka nchekwa, bufee supernatant na tube centrifuge ọzọ na-adịghị mma ma chekwaa na -20℃ maka izu 2.

2.PCR Amplification

Wepu 2 × Mouse Tissue Direct PCR Mix si -20 ℃ wee mee ka ọ daa na ice, gwakọta ihu ọma wee kwadebe usoro mmeghachi omume PCR dị ka tebụl na-esonụ (na-arụ ọrụ na ice):

Mara:

a) Ọnụ ego agbakwunyere ekwesịghị gafere 1/10 nke usoro ahụ, ma ọ bụrụ na agbakwunyere nke ukwuu, enwere ike igbochi PCR amplification.

Ọnọdụ PCR akwadoro

Mara:

a) Annealing okpomọkụ: N'iburu n'uche na Tm uru nke primer, a na-atụ aro ka ịtọ okpomọkụ annealing na nta Tm uru nke primer +3 ~ 5 ℃.

Nsogbu na ngwọta nkịtị

1.Enweghị ibe ezubere iche

1) Ngwaahịa lysis gabigara ókè.Họrọ ndebiri nke kacha kwesị ekwesị, na-adịkarịghị karịa 1/10 nke sistemụ;

2) Oke sample size.Ghichaa lysate ugboro 10 wee gbasaa, ma ọ bụ belata nha nlele na re-lysis;

3) Tissu samples adịghị ọhụrụ.A na-atụ aro ka iji ihe nlele anụ ahụ ọhụrụ;

4) Ogbenye primer àgwà.Jiri DNA genomic maka mmụba iji nyochaa ịdịmma primer ma kwalite nhazi nke mbụ.

2.Nkwalite na-abụghị nke akọwapụtara

1) Okpomọkụ na-ekpo ọkụ dị oke ala na ọnụọgụ okirikiri dị elu.Mee ka okpomọkụ na-egbuke egbuke ma belata ọnụ ọgụgụ nke okirikiri;

2) Ntinye uche nke ndebiri dị oke elu.Belata ego nke ndebiri ma ọ bụ megharịa ndebiri ugboro 10 mgbe nkwalite;

3) Nkọwapụta primer adịghị mma.Kwalite nhazi ọkwa mbụ.

Ihe ndetu

1.Iji zere mmetọ obe n'etiti ihe nlele, a ga-edozi ọtụtụ ngwaọrụ nlele, na enwere ike iji 2% sodium hypochlorite solution ma ọ bụ ihe nchacha nucleic acid sachaa elu ngwaọrụ mgbe nlele ọ bụla ma ọ bụrụ na achọrọ ya ugboro ugboro.

2.A na-atụ aro ka iji anụ ahụ òké ọhụrụ, na olu nlele ekwesịghị ịdị ukwuu iji zere imetụta nsonaazụ nkwalite.

3.Lysis Buffer kwesịrị ịzenarị friza ugboro ugboro, yana enwere ike ịchekwa ya na 2 ~ 8 ℃ maka ojiji dị mkpirikpi.Ọ bụrụ na echekwara ya na obere okpomọkụ, mmiri ozuzo nwere ike ime, na a ga-agbaze nke ọma tupu ejiri ya.

4.Ngwakọta PCR kwesịrị ịzenarị friza ugboro ugboro, enwere ike ịchekwa ya na 4 ℃ maka iji obere oge mee ihe ugboro ugboro.

5.Ngwaahịa a bụ naanị maka nyocha nnwale sayensị na ekwesighi iji ya na nyocha ụlọ ọgwụ ma ọ bụ ọgwụgwọ.