Ngwa Methylation-specipc PCR (MSP)

Ngwa nchọpụta PCR nke methylation akọwapụtara.

A na-emepụta Kit PC-MSR (MSP) nke methylation-ọpụrụiche maka ndị ahịa na-amụ njirimara methylation nke DNA genomic site na PCR, nke MSP DNA Polymerase bụ polymerase thermostable gbanwere na ọgwụ mgbochi, yana 10 × MSP PCR Buffer bụ ihe nchekwa PCR kachasị maka Mmeghachi omume MSP. Ọ dakọtara na Kit Nchigharị Bisulfite DNA TIANGEN (4992447).

Nwamba. Mba Nkwakọ ngwaahịa
4992759 50 ngụkọta ọnụ

Nkọwa ngwaahịa

Usoro ọrụ

Ihe Nlereanya Nnwale

Ajụjụ

Mkpado ngwaahịa

Atụmatụ

■ Ngwaahịa nwere uru nke ngwa ngwa, ịdị mfe, ịdị elu dị elu, nkọwapụta siri ike na nkwụsi ike dị mma.

Nkọwapụta

Ụdị: Polymerase nke DNA MSP
Ụkpụrụ: <500 ng
Ngwa: Ọ dabara maka usoro PCR (MSP) akọwapụtara methylation iji nyochaa njirimara methylation nke DNA genomic

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


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    1. Ejiri ngwa PCR (MSP) methylation-iji kọwaa iberibe bp 400 site na iji bisulfite mesoo DNA genomic dị ka ndebiri nwere sistemụ mmeghachi omume nke 20 μl.

    Experimental Exampl

    2. Ntọala okirikiri mmeghachi omume PCR

    Experimental Exampl

     

    Experimental Examples 1: Nlele nlele nke onye na -eweta A;
    2: Nlele nke onye na -ebunye B na -ahazi;
    3: TIANGEN mesoo sample 4: NTC; M: D2000
    Experimental Examples 1-6:
    6 na -emegharị;
    7: Onye na -eweta ngwaahịa A haziri nlele;
    8: NTC; Bio2-F/R na P16- Me-F/R: Primers nchọpụta abụọ.
    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?
    ytry
    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.

    Dee ozi gị ebe a ma zitere anyị ya