Ngwa PCR ozugbo osisi

Mbambamba: HCR2020A

Plant Direct PCR Kit kwesịrị ekwesị maka mmụba ozugbo nke akwụkwọ osisi, mkpụrụ osisi, wdg, enwere ike iji ya mee nyocha ihe nlele dị elu nke ihe ọkụkụ na-enweghị polysaccharides na polyphenols.Nkwalite DNA polymerase kpọmkwem dabere na evolushọn eduzi nwere nnabata dị elu nye ndị na-egbochi PCR na osisi.Ka ọ dị ugbu a, ọ na-ejigide arụmọrụ mmụba dị elu, nke kwesịrị ekwesị maka mmụba nke iberibe DNA n'ime 5 kb.Enwere ike iji ihe nchekwa Lysis pụrụ iche A dị n'ime ngwa wee lyse anụ ahụ osisi ọhụrụ ma ọ bụ oyi kpọnwụrụ.Ọ dị mfe ịrụ ọrụ na lysate nwere ike iji mee ihe dị ka template maka mmụba na-enweghị ọcha.Sistemu ahụ nwere ihe nchebe nke na-enyere aka ịgbatị ihe nlele crude nke ọma mgbe ịsacha na agbaze ugboro ugboro.2 × Plant Direct Master Mix naanị kwesịrị ịgbakwunye primers na ndebiri iji mee mmeghachi omume mmụba, si otú ahụ belata ọrụ pipetting na melite mmepụta mmepụta na reproducibility nke nsonaazụ.

Ngwa

* Plant Direct Lysis Buffer B bụ nhọrọ reagent, nke a na-eji na-ewepụ Plant Direct Lysis Buffer A maka ịgbatị oge nchekwa nke ihe nlele.Enwere ike iji ya dabere na ọnọdụ ahụ n'ezie.

Ọnọdụ Nchekwa

2 × Plant Direct Master Mix, na-echekwa na -30 ~ -15 ℃ ma zere ịfriza ugboro ugboro na thawing;Plant Direct Lysis Buffer, na-echekwa na -30 ~ -15℃ ma ọ bụ 2 ~ 8℃.

Usoro nnwale

Nhazi ihe nlele-Akwụkwọ osisi

Usoro ozugbo:A na-atụ aro ka iji akwụkwọ na-eto eto.Jiri oghere oghere nke nwere dayameta nke 0.5 - 3 mm iji nweta obere ihe atụ na otu, wee tinye ozugbo na usoro PCR (usoro 50 μl na-atụ aro).Rịba ama, jide n'aka na ihe nlele ahụ dị na ngwọta PCR ọ bụghị megide mgbidi tube.Ọ bụrụ na a na-eji PCR kpọmkwem mee ka mpekere dị ogologo na ihe nlele dị mgbagwoju anya, iji ihe nlele nwere obere dayameta (0.5 - 1 mm) dịka ndebiri nwere ike inye aka nweta nsonaazụ ka mma.

Usoro lysis egweri:A na-atụ aro ka iji akwụkwọ na-eto eto.Were obere mpempe akwụkwọ (ihe dị ka 1 - 3 mm na dayameta), tinye ya na 20 μl Plant Direct Lysis Buffer Ab, ma gwerie ya dị ka o kwere mee (enwere ike ime nzọụkwụ a site na ịpịnye akwụkwọ ahụ na 100 μl pipette n'ọnụ. iji mebie sample).Ọ bụrụ na ejiri mpempe akwụkwọ buru ibu (agaghị agafe 7 mm), mee ka ụda dilution dị elu ruo 50 μl.Mgbe akwụkwọ ahụ gwụchara, ihe ngwọta kwesịrị ịpụta akwụkwọ ndụ akwụkwọ ndụ.Mgbe obere centrifugation gachara, tinye 1 μl nke supernatant na sistemụ PCR dị ka ndebiri mmeghachi omume.

Usoro nke thermal lysis:A na-atụ aro ka iji akwụkwọ na-eto eto.Were obere mpempe akwụkwọ (ihe dịka 1 - 3 mm na dayameta), tinye ya na 20 μl Plant Direct Lysis Buffer A, ma kpoo ya na 95 ° C maka 5 - 10 min.Enwere ike ịgbatị oge lysis nke ọma maka akwụkwọ ndị siri ike lyse (ọ bụghị ihe karịrị 20 min).Ọ bụrụ na ejiri mpempe akwụkwọ buru ibu (agaghị agafe 7 mm), mee ka ụda dilution dị elu ruo 50 μl.Mgbe kpochara, centrifuge obere oge, ma tinye 1 μl nke supernatant na sistemụ PCR dị ka ndebiri mmeghachi omume.

Nhazi ihe nlele–Mkpụrụ osisi

Usoro lysis egweri:Jiri pensụl gbutu mkpụrụ nke nwere dayameta nke 5 mm, tinye ya na 100 μl Plant Direct Lysis Buffer A, ma jiri ntu pipette ma ọ bụ ngwaọrụ ndị ọzọ gwerie sample.Vortex dị nkenke ma hapụ ya ka ọ guzoro na ụlọ okpomọkụ maka 3 - 5 min.Jide n'aka na ihe nlele nke mkpụrụ ahụ na-emikpu n'ime ihe nkpuchi mmiri.Mgbe obere centrifugation gachara, tinye 1 μl nke supernatant na sistemụ PCR dị ka ndebiri mmeghachi omume.

Usoro nke thermal lysis:Jiri pensụl egbutu mkpụrụ nke nwere dayameta nke 5 mm, tinye ha na 100 μl Plant Direct Lysis Buffer A, na ikpo ọkụ na 95 Celsius C maka 5 - 10 min.Enwere ike ịgbatị oge lysis nke ọma maka akwụkwọ ndị siri ike lyse (ọ bụghị ihe karịrị 30 min).Mgbe kpochara, centrifuge obere oge, ma tinye 1 μl supernatant na sistemụ PCR dị ka ndebiri mmeghachi omume.

a.A pụkwara iji mkpasa ma ọ bụ ngwá ọrụ ndị ọzọ na-egbutu ihe atụ nke nha kwesịrị ekwesị;Ọ bụrụ na ejigharịgharị ntụmadị ma ọ bụ mkpa ahụ, a ga-ehichapụ ha na ngwọta 2% sodium hypochlorite tupu ejiri nke ọ bụla mee ihe iji gbochie mmetọ n'etiti ihe nlele.

b.Gbaa mbọ hụ na ihe nchekwa Plant Direct Lysis na-agbaze nke ọma tupu eji ya.Ọ bụrụ na ihe nchekwa ahụ bụ viscous ma ọ bụ nwere precipitates, ọ nwere ike ikpo ọkụ na 37 ℃ iji gbazee ya kpamkpam tupu ejiri ya.

c.Enwere ike imezi olu nke template na usoro mmeghachi omume n'ụzọ kwesịrị ekwesị dị ka ọdịiche dị na olu nke ihe ọkụkụ na diluent gbakwunyere.

Osisi Direct Lysis nchekwa

The Plant Direct Lysis Buffer A nke dị na ngwaahịa a ka emebere nke ọma ịhapụ genome nke ọtụtụ anụ ahụ osisi ma dabara adaba maka nchekwa obere oge nke osisi crude na 4 ℃.Ọ bụrụ na ekwesịrị ịchekwa ihe nlele ahụ ogologo oge (dịka ọmụmaatụ, ọnwa 1-2), a na-atụ aro ka ịnyefe supernatant na tube EP ọhụrụ ma chekwaa ya na -20 ℃.Iji chekwaa ihe nlele ndị ahụ nke ọma, tinye nha nha nha nke Plant Direct Lysis Buffer B na nnukwu ihe ebufere, gwakọta nke ọma ma chekwaa na -20 ℃.Oge nchekwa kwụsiri ike dị iche na ihe atụ osisi na steeti.

Sistemụ mmeghachi omume

a.O nwere mg²⁺na njedebe ikpeazụ nke 2 mmM.

b.A na-atụ aro ka iji ntinye ikpeazụ nke 0.4μM maka primer ọ bụla.Iji primers emebiga ihe ókè ga-eduga n'ịbawanye mmụba na-enweghị isi.

c.Enwere ike ịhazi ọnụọgụ nlele ejiri mee ihe dịka ọnọdụ dị adị.Enwere ike gbanwee ego ejiri mee otu mmeghachi omume nke ihe nlele lysed crude n'etiti 2% - 20% nke mkpokọta mmeghachi omume.Iji ọtụtụ ihe nlele nwere ike ibute ọdịda nkwuputa.

Mmemme mmeghachi omume

a.Denaturation mbụ (98 ℃, 5 min) na-akwalite lysis nke anụ ahụ osisi, na-ahapụ DNA genomic nke enwere ike iji maka mmụba PCR.Emela ka oge dị mkpụmkpụ ma ọ bụ belata okpomọkụ.

b.A na-atụ aro ka ịtọ ya ka uru Tm primer ma ọ bụ 2 ~ 4 ℃ dị elu karịa uru Tm.Nkwalite DNA polymerase nke ejiri na ngwaahịa a dị iche na nke Taq DNA polymerase nke a na-ahụkarị, ma nwee ihe pụrụ iche chọrọ maka mmeghachi omume na-ekpo ọkụ; iji okpomọkụ na-ekpo ọkụ dị elu nwere ike belata mmụba na-enweghị isi ma melite arụmọrụ nkwalite ahụ.Maka ndebiri mgbagwoju anya, enwere ike nweta nkwalite ahụ nke ọma site n'ịgbanwe ọnọdụ okpomọkụ na ịgbatị oge mgbatị.

c.Ọ bụrụ na ogologo ngwaahịa nkwalite bụ ≤1 kb, a na-etinye oge mgbatị ahụ na 30 sec / kb;ọ bụrụ na ogologo ngwaahịa nkwalite bụ> 1 kb, a na-edozi oge mgbatị ahụ na 60 sec / kb.

d.Maka ihe nlere anya dị mgbagwoju anya ma ọ bụ ihe nlele nwere obere mmụba, ọnụ ọgụgụ nke okirikiri nwere ike ịbawanye nke ọma na okirikiri 40 -50.

Ngwa

Ọ na-adabara maka mmụba ozugbo nke anụ ahụ osisi na nyocha dị elu nke ihe atụ nke ihe ọkụkụ na-enweghị polysaccharides na polyphenols.

Ihe ndetu

Maka iji naanị nyocha.Ọ bụghị maka ojiji na usoro nyocha.

1. Maka mmụba osisi crude ma ọ bụ nkwalite kpọmkwem, a na-atụ aro ka iji DNA genomic dị ọcha dị ka njikwa dị mma tupu ịmalite nyocha ahụ iji hụ na usoro, primers na arụmọrụ ziri ezi.

2. Nkwalite DNA polymerase kpọmkwem nke ejiri na ngwa a nwere ọrụ nyocha siri ike.Ọ bụrụ na ọ dị mkpa ka a rụọ cloning TA, a na-atụ aro ka ọ dị ọcha DNA tupu ị gbakwunye adenine.

3. Nduzi Ntuzi Nhazi:

a.A na-atụ aro na ntọala ikpeazụ na njedebe 3′ nke primer kwesịrị ịbụ G ma ọ bụ C.

b.Ekwesịrị izere ndakọrịta na-esoghị na ntọala 8 ikpeazụ na njedebe 3′ nke primer.c.Zere nhazi ntutu isi na njedebe 3′ nke primer.

d.Ọdịiche dị na Tm uru nke n'ihu primer na reverse primer ekwesịghị ịbụ ihe karịrị 1 ℃ na Tm uru kwesịrị ịgbanwe ka 60 ~ 72 ℃ (Primer Premier 5 na-atụ aro ka gbakọọ Tm uru).

e.Usoro mgbakwunye agbakwunyere nke na-adakọghị na ndebiri, ekwesighi ịgụnye mgbe ị na-agbakọ uru Tm primer.

f.A na-atụ aro ka ọdịnaya GC nke primer bụrụ 40% -60%.

g.Nkesa n'ozuzu nke A, G, C na T na primer kwesịrị ịdị ka o kwere mee.Zere iji mpaghara nwere nnukwu ọdịnaya GC ma ọ bụ AT.

h.Zere ọnụnọ nke usoro nkwado nke 5 ma ọ bụ karịa ntọala ma n'ime primer ma ọ bụ n'etiti primer abụọ ma zere ọnụnọ nke usoro nkwado nke 3 ma ọ bụ karịa na njedebe 3′ nke primers abụọ.

i.Jiri ọrụ NCBI BLAST iji lelee nkọwapụta nke primer iji gbochie mmụba na-enweghị isi.