2 Mix Ngwakọta Pfu PCR

Ezigbo ntụkwasị obi dị elu Taq DNA polymerase.

E gosipụtara Pfu DNA Polymerase site na E.coli na mkpụrụedemede Pyrococus Furiosis DNA Polymerase ma mee ka ọ dị ọcha ma kewapụ ya site na ọtụtụ nsacha kọlụm. N'ihi na Pfu nwere ọrụ exonuclease 3′-5,, ọ nwere ike nyochagharị na usoro nkwalite DNA, ebe ọdịnala Taq DNA Polymerase enweghị ike. Ọ bụ ezie na polymerases Taq DNA ndị ọzọ dị ka Vent, Deep Vent, Tli, UITma, wdg nwere ọrụ ịtụgharị ihe, Pfu nwere ọnụego adịghị ala kacha nta n'etiti polymerases Taq DNA niile achọtara. Pfu DNA Polymerase nwere nkwụsi ike ọkụ ka mma karịa Taq DNA polymerase, ọ nwere ike idowe ihe karịrị 90% ọrụ na 95 ° C maka elekere 1.

Otu-tube Pfu PCR Ngwakọta (Asambodo Ngwa Ngwa Ọha nke Mba)

Ngwakọta Pfu PCR emeziwanye nkọwapụta na mmetụta nke mmeghachi omume PCR ma nwee ike bulie ndebiri dị mgbagwoju anya yana ọdịnaya GC dị elu, usoro nke abụọ na ihe ndị yiri ya. Enwere ike ịgbasawanye dị ka mbipụta 2 nke ndebiri ebumnuche, na -eme ka nnwale nnwale dabara adaba.

Formula Usoro Pfu MasterMix pụrụ iche na-eme ka usoro mmeghachi omume niile kwụsie ike, na friza ma ọ bụ nchekwa ogologo oge na 4 Celsius agaghị emetụta ọrụ ahụ.

Solution Ngwọta ngwakọta PCR kwụsiri ike ma kwadebere nke ọma nwere ike ime ka ọrụ ahụ dị ngwa ma dịkwa mfe, na-ebelata oke ọrụ na njehie nlele. A na-etinye nkwalite PCR dị elu na ihe nrụpụta dị elu na ngwakọta, nke na-ebelata ihe achọrọ na ọnọdụ PCR.

■ Ngwaahịa a nwere sistemụ nwere nke nwere agba na agba. Ngwaahịa PCR Mix nwere agba nwere ike bụrụ electrophoresed ozugbo PCR gachara, na-etinyeghị ihe nlele nlele.

Nwamba. Mba	Nkwakọ ngwaahịa
4992780	1ml
4992781	5*1ml
4992782	1ml
4992906	5*1ml

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Nkọwa ngwaahịa

Usoro ọrụ

Ajụjụ

Mkpado ngwaahịa

Nkọwa ọrụ

A kọwapụtara ọrụ nke otu (U) Pfu DNA Polymerase dị ka ọnụ ọgụgụ enzyme achọrọ iji tinye 10 nmol deoxynucleotides n'ime ihe ndị na-anaghị agbaze acid na 74 Celsius C n'ime nkeji iri atọ site na iji DNA sperm DNA arụ ọrụ dị ka ndebiri/mbido.

Njikwa Ogo

Ịdị ọcha site na nchọpụta SDS-PAGE karịrị 99%; Achọpụtaghị ọrụ nke nuclease exogenous; Enwere ike ịbawanye otu mkpụrụ ndụ ihe nketa na mkpụrụ ndụ ihe nketa mmadụ nke ọma; Enweghị mgbanwe ọrụ dị mkpa mgbe echekwara ya n'ime ụlọ okpomọkụ otu izu.

Ngalaba Nka na ụzụ

Ọ nwere ọrụ exouclease 3′-5 and na enweghị ọrụ ọpụpụ 5′-3.. Mgbatị ndọtị nke nkwalite DNA dị ala karịa nke Taq polymerase, na n'ozuzu mgbatị mgbatị nke enfu Pfu bụ 0.5-1 kb kwa nkeji. Nkwụsi ike nke Pfu dị mma karịa Taq. Maka ndebiri nwere ọdịnaya GC dị elu, enwere ike ịbawanye okpomọkụ denaturation ruo 98 Celsius, nke na -enweghị mmetụta na ọrụ Pfu polymerase. Ngwa PCR agwụla nke ọma, nke enwere ike ịgbakwunye ya na 3'-dA overhags tupu ejikọ ya na vector TA ma ọ bụ kpuchie ya na vector ngwụcha.

Ngwa

Enwere ike iji ya maka nkwalite ntụkwasị obi dị elu nke DNA, dị ka cloning gene gene, mutation na-eduzi saịtị, nyocha otu nucleotide polymorphism (SNP) na mmezi njedebe.

Enwere ike ịhazi ngwaahịa niile maka ODM/OEM. Maka nkọwa,biko pịa Ọrụ ahaziri (ODM/OEM)

Nke gara aga: Ngwa Kitag Mutagenesis na-eduzi ngwa ngwa

Osote: Ultra HiFidelity PCR Kit

Jiri DNA genomic dị ka ndebiri iji bulie ibe 1kb.
Mgbe mmeghachi omume PCR, were 5 μl maka nchọpụta electrophoresis.

Ajụjụ: Enweghị ìgwè mmụba

Ụkpụrụ A-1

Template nwere adịghị ọcha protein ma ọ bụ ndị na -egbochi Taq, wdg.

■ Mmebi nke ndebiri ahụ ezubeghị ——- Mee ka okpomoku denaturation mụbaa nke ọma ma gbatịkwuo oge denaturation.

■ Mbibi nke ndebiri ——Ghazigharịa ndebiri ahụ.

A-2 Primer

Quality Ogo adịghị mma nke pimerị — —Re-synthesize primer.

Mbibi Primer ——Na -ebuli isi ihe dị elu n'ime obere olu maka ichekwa. Zere ọtụtụ ntu oyi na ịzeze ma ọ bụ chekwaa ogologo oge ruo 4 Celsius.

Design Ọdịdị na -adịghị mma nke primer (dịka ọmụmaatụ, ogologo oge ezughi ezu, dimer etolite n'etiti ihe nrịbama, wdg) -Pịgharịa ihe nrụpụta (zere imepụta primer dimer na nhazi nke abụọ)

A-3 mg²⁺ịta

■ mg²⁺ ịta ahụhụ pere mpe ——Mụba Mg nke ọma²⁺ịta: kwalite Mg²⁺ itinye uche site na ọtụtụ mmeghachi omume sitere na 1 mM ruo 3 mM nwere nkeji 0.5 mM iji chọpụta ezigbo Mg²⁺ ịta maka ndebiri na mbido ọ bụla.

A-4 Okpomoku na-agwụ ike

■ Okpomoku dị elu na -emetụta njikọta nke mbido na ndebiri. ——Mbelata okpomọkụ na -agba ume ma bulie ọnọdụ ya na gradient nke 2 Celsius.

A-5 Mgbakwunye oge

Time Oge ịgbatị dị mkpirikpi —— Mụbaa oge ndọtị.

Ajụjụ: ọ dị mma

Phenomena: Ihe nlele na -adịghị mma na -egosikwa ụgbụ usoro ebumnuche.

A-1 mmetọ nke PCR

Gafee nrubeisi nke usoro ebumnuche ma ọ bụ ngwaahịa nkwalite ——Kpachapụ anya ka ị ghara ịwụnye ihe nlele nwere usoro lekwasịrị na nlele na -adịghị mma ma ọ bụ wụpụ ya na tube centrifuge. Ekwesịrị idobe ihe reagents ma ọ bụ akụrụngwa iji kpochapụ acid nucleic dị adị, ekwesịrị ikpebi ịdị adị nke mmetọ site na nnwale nchịkwa ọjọọ.

Contamin Mmetọ na -emegharị ihe——Nyefe ihe reagent ma chekwaa na obere okpomọkụ.

A-2 Primer

■ mg²⁺ ịta ahụhụ pere mpe ——Mụba Mg nke ọma²⁺ ịta: kwalite Mg²⁺ itinye uche site na ọtụtụ mmeghachi omume sitere na 1 mM ruo 3 mM nwere nkeji 0.5 mM iji chọpụta ezigbo Mg²⁺ ịta maka ndebiri na mbido ọ bụla.

Nhazi mmalite adịghị mma, yana usoro ebumnuche lekwasịrị anya nwere usoro na-abụghị ebumnuche. ——Rer-design primers.

Ajụjụ: Nkọwapụtaghị nke akọwapụtara

Phenomena: Mgbakwunye nkwalite PCR adabaghị na nha a tụrụ anya ya, ma ọ bụ buru ibu ma ọ bụ obere, ma ọ bụ oge ụfọdụ otu mgbatị ahụ akọwapụtara nke ọma na otu mmụba na-abụghị nke akọwapụtara na-eme.

A-1 Primer

Specific Nkọwapụta adịghị mma

——Re-design primer.

Ọdịmma nke mbido dị elu nke ukwuu ——Mụbawanye okpomoku denaturation ma gbatịkwuo oge denaturation.

A-2 mg²⁺ ịta

M The Mg²⁺ ịta ahụhụ dị elu nke ukwuu ——Hụlata mgbatị Mg2+ nke ọma: kwalite Mg²⁺ itinye uche site na ọtụtụ mmeghachi omume sitere na 1 mM ruo 3 mM nwere nkeji 0.5 mM iji chọpụta ezigbo Mg²⁺ ịta maka ndebiri na mbido ọ bụla.

A-3 polymerase na-enweghị ike

Amount Oke enzyme nke ukwuu ——Bụlata ego enzyme n'ụzọ dabara adaba na etiti 0.5 U.

A-4 Okpomoku na-agwụ ike

■ Okpomoku annealing adịchaghị ala——- Kwesịrị iwelie okpomọkụ annealing n'ụzọ kwesịrị ekwesị ma ọ bụ were usoro nku abụọ.

A-5 PCR okirikiri

Cy Ọtụtụ okirikiri PCR —— Belata ọnụọgụ okirikiri PCR.

Ajụjụ

A-1 Primer——Ọdịdị adịghị mma ——Tụgharịa ihe nrụpụta ihe, gbanwee ọnọdụ na ogologo nke ihe mbido iji mee ka nkọwa ya ka mma; ma ọ bụ mee PCR akwụkwụ.

DNA Template A-2

——Mpempe akwụkwọ ahụ adịghị ọcha ——Kachapụ ndebiri ahụ ma ọ bụ wepụta DNA ya na ngwa ncha.

A-3 mg²⁺ ịta

——Mg²⁺ ịta ahụhụ dị oke elu --—Hazie Mg²⁺ ịta: kwalite Mg²⁺ itinye uche site na ọtụtụ mmeghachi omume sitere na 1 mM ruo 3 mM nwere nkeji 0.5 mM iji chọpụta ezigbo Mg²⁺ ịta maka ndebiri na mbido ọ bụla.

A-4 dNTP

——Ndị njupụta nke dNTP dị oke elu—— Belata itinye uche nke dNTP n'ụzọ dabara adaba

A-5 Okpomoku na-agbakwụnye

——Obere okpomọkụ annealing dị ala—— Mee ka ọkụ annealing dịkwuo mma

Cycles A-6

——Otutu okirikiri ——Bụgharịa ọnụọgụ okirikiri

Ajụjụ: Ego ole ka ekwesịrị ịgbakwunye na sistemụ mmeghachi omume 50 PCl PCR?

Ajụjụ: Kedu ka ị ga -esi gbasaa ogologo iberibe?

Nzọụkwụ mbụ bụ ịhọrọ polymerase kwesịrị ekwesị. Tame polymerase oge niile enweghị ike ịgụgharị ya n'ihi enweghị ọrụ exouclease 3'-5 ', na enweghị nkwekọrịta ga-ebelata arụmọrụ ndọtị nke iberibe. Yabụ, Taq polymerase oge niile enweghị ike ịgbasa iberibe ebumnuche ezubere karịa 5 kb. A ga -ahọrọ Taq polymerase nwere mgbanwe pụrụ iche ma ọ bụ polymerase ọzọ dị elu iji melite arụmọrụ ndọtị yana gboo mkpa nkwalite ogologo nkenke. Na mgbakwunye, mmụba nke iberibe ogologo na-achọkwa mmezi kwekọrọ ekwekọ nke ihe mbido, oge denaturation, oge mgbatị, pH na-echekwa, wdg. Iji gbochie mmebi ndebiri, oge denaturation na 94 Celsius ga -agbada na 30 sec ma ọ bụ obere oge okirikiri, na oge ịrị elu okpomọkụ ruo 94 Celsius C tupu mmụba ga -erughị 1 min. Ọzọkwa, ịtọlite ọnọdụ mgbatị ahụ n'ihe dị ka 68 Celsius na ịhazi oge mgbatị ahụ dị ka ọnụego 1 kb/min nwere ike hụ na ọ na -abawanye nke ọma nke iberibe ogologo.

Ajụjụ: Kedu ka esi kwalite ntụkwasị obi nke PCR?

Enwere ike belata ọnụego njehie nke nkwalite PCR site na iji polymerases DNA dị iche iche nwere ezigbo ntụkwasị obi. N'etiti polymerases Taq DNA niile achọtara ruo ugbu a, enzyme Pfu nwere ọnụego mmejọ kachasị dị ala na ntụkwasị obi kachasị elu (lee okpokoro agbakwunyere). Na mgbakwunye na nhọrọ enzyme, ndị ọrụ nyocha nwere ike belata mbelata PCR site na ime ka ọnọdụ mmeghachi omume dị mma, gụnyere imepụta ngwongwo nchekwa, itinye polymerase kachasị mma na ime ka ọnụọgụ okirikiri PCR.

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