Kit Kit PCR Ọbara

Mgbasa ngwa ngwa nke mkpụrụ ndụ ihe nketa a na -ele anya na -eji ọbara dị ka ndebiri na -enweghị mmịpụta.

Ngwa a na-anabata ihe mgbochi ihe mgbochi DNA polymerase ka ọ gbasaa nke ọma otu mkpụrụ ndụ ihe nketa na mkpụrụ ndụ ihe nketa mmadụ. Sistemụ nchekwa dị mma nke ọma na ngwa a na -enyere polymerase aka iguzogide mgbochi nke ndị na -egbochi PCR, yabụ nwere ike gbasaa DNA ozugbo site na iji ọbara na mkpụrụ ndụ emebere emezi dị ka ndebiri. Ngwaahịa a dị mfe ịrụ ọrụ, ọ chọghịkwa usoro dị mgbagwoju anya dịka nsacha DNA ma ọ bụ nlele nlele.
Enyere ngwa a ka ọ bụrụ 2 × MasterMix, enwere ike ịme mmeghachi omume ahụ site na ịtinye ndebiri ọbara yana ihe nchoputa kwekọrọ. Enwere ike itinye ya na sel nke anụ mammals dị ka mmadụ, òké, ezì, ehi na ụdị ndị ọzọ, yana ọbara zuru oke ma ọ bụ 4 ℃ cryopreserved, anticoagulant (EDTA, citrate, heparin), mkpụkọ ọbara na -egbu egbu na ntụpọ ọbara. echekwara na Whatman 903 na FTA Elute kaadị azụmahịa.

Nwamba. Mba	Nkwakọ ngwaahịa
4992529	20 µl × 100 rxn
4992530	20 µl × 500 rxn

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Nkọwa ngwaahịa

Nlereanya Nnwale

Ajụjụ

Mkpado ngwaahịa

Atụmatụ

■ Dị Mfe na ngwa ngwa: Enwere ike ịme nkwalite PCR ozugbo site na iji ọbara dị ka ndebiri, na -enweghị mkpa usoro siri ike nke nkwadebe nlele na mmịpụta DNA.
Urity Ịdị ọcha dị elu: Ibefe nlele nlele nlele yana usoro mmịpụta DNA nwere ike inyere aka izere mmetọ nke nlele.
Nsonaazụ dị elu: Enwere ike ịme njirimara PCR maka nlele buru ibu site na ijikọ kit na efere PCR 96/384.
Ọdịdị zuru ụwa ọnụ: Ngwa a nwere ike ime ka iberibe GC dị elu ma ọ bụ iberibe nwere usoro nke abụọ dị mgbagwoju anya, ogologo nkwalite nwere ike iru 5 kb.
Resistance Nrụgide nrụgide siri ike: Enwere ike itinye ngwa a maka ụdị dị iche iche na nlele ọbara echekwara n'ụzọ dị iche iche.

Ngwa

Ngwa PCR nke ngwa a nwere “A” na njedebe 3′, nke enwere ike iji ya ozugbo maka mkpuchi vector TA. Enwere ike iji ngwa a maka mmụba nke iberibe DNA genomic, nyocha mkpụrụ ndụ ihe nketa dị elu na nyocha genotyping (dịka nchọpụta mkpụrụ ndụ).

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

Nke gara aga: TIANcombi DNA Lyse & Det PCR Kit

Osote: Ngwa mpempe akwụkwọ PCR ngwa ngwa

	N'iji anticoagulation EDTA mmadụ dị ka ndebiri, mkpụrụ ndụ ihe nketa 4 nwere ọdịnaya GC dị iche iche gbalitere site na Kit Direct PCR. Usoro mmeghachi omume PCR bụ 20 μl, ejiri ọbara 1 μl mee ihe dị ka ndebiri. Nna -ukwu: TIANGEN akara 2; 1: Iberibe nha 1090 bp, ọdịnaya GC 68.1%; 2: Iberibe nha 1915 bp, ọdịnaya GC 70.4%; 3: Ogo ibe 448 bp, ọdịnaya GC 74.8%; 4: Ogo ibe 1527 bp, ọdịnaya GC 61.5%. Nsonaazụ nnwale: Kit Direct PCR nke Ọbara nwere ike ịbawanye iberibe DNA yana ọdịnaya GC n'etiti 61.5%-74.8%, na-atụ aro na ọ nwere ike ịbawanye iberibe GC dị elu.
	N'iji anticoagulation EDTA mmadụ dị ka ndebiri, mkpụrụ ndụ ihe nketa 5 nwere ogologo dị iche iche (ActB, Prp, DN1.0, Hn2.0 na Hn4.0) gbalitere site na Kit Direct PCR. Usoro mmeghachi omume PCR bụ 20 μl, ejiri ọbara 1 μl mee ihe dị ka ndebiri. Nna -ukwu: TIANGEN akara 2; 1-3: 3 nlele ọbara dị iche iche; NTC: njikwa na -enweghị primers. Nsonaazụ nnwale: Kit Direct PCR nke Ọbara nwere ike ime ka mpekere dị ogologo ruo 4 kb, na -atụ aro na ọ nwere ike ịbawanye iberibe ogologo.
	N'iji anticoagulation EDTA mmadụ dị ka ndebiri, ejiri PCR Kit nke Ọbara mee nchọpụta PCR nke ụdị ọbara dị iche iche. Usoro mmeghachi omume PCR bụ 20 μl, ejiri ọbara 1 μl mee ihe dị ka ndebiri. Nna -ukwu: TIANGEN akara 2; 1-9: oke ibu ọbara bụ 0.1 μl, 0.2 μl, 0.3 μl, 0.4 μl, 1 μl, 2 μl, 3 μl, 4 μl na 5 μl, n'otu n'otu; NTC: jikwaa na enweghị ndebiri Nsonaazụ nnwale: Kit Direct PCR nke ọbara nwere nguzogide siri ike na ọbara ma nwee ike gbasaa nlele ọbara site na ntinye nke 0.1-5 μl.
	Ejiri nlele ọbara sitere na mmadụ, oke, ọkụkọ na ụdị ndị ọzọ nwere ọgwụgwọ dị iche iche dị ka ndebiri. Ejiri ngwa PCR nke Ọbara gbasawanye PRNP (mmadụ, 750 bp), Actin (oke, 200 bp), na β-Actin (Chicken, 1.0 kb). Usoro mmeghachi omume PCR bụ 20 μl, ejiri ọbara 1 μl mee ihe dị ka ndebiri. Nna -ukwu: TIANGEN Marker II. Nsonaazụ nnwale: Enwere ike itinye Kit PCR Blood Direct n'ọtụtụ nlele dị iche iche, enwere ike ime nchọpụta PCR ozugbo na nlele ọbara sitere n'ụdị dị iche iche nwere ọgwụgwọ dị iche iche.

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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