Ngwa PCR ọtụtụ

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.

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.

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.

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

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.

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.