Ekwenyere m na onye ọ bụla ga-ezute nsogbu ndị dị otú ahụ ma ọ bụ ndị dị otú ahụ mgbe ọ na-eme mmeghachi omume PCR, ma ọtụtụ n'ime ha nwere ike kewaa n'ime isi nsogbu abụọ:

Nkwalite obere mkpụrụ ndụ ihe nketa (ịkwalite);
Mmụba mkpụrụ ndụ ihe nke na-abụghị ebumnuche.
Iji ihe mgbakwunye bụ otu n'ime atụmatụ a na-ejikarị edozi nsogbu ndị a.Ọtụtụ mgbe, ọrụ nke mgbakwunye nwere akụkụ abụọ:
usoro nke abụọnke mkpụrụ ndụ ihe nketa (usoro nke abụọ);
Belata priming na-abụghị nke akọwapụtara.
Taa, onye nchịkọta akụkọ ga-ewebata gị nkenke ihe mgbakwunye ndị a na-ahụkarị na mmeghachi omume PCR na ọrụ ha.
Ihe mgbakwunye na-ebelata usoro nke abụọ
sulfoxide(DMSO)
mkpụrụ ndụ ihe nketanwere nnukwu ọdịnaya GC.Agbanyeghị, DMSO na-ebelatakwa ọrụ Taq polymerase nke ukwuu.Ya mere, onye ọ bụla ga-edozi ohere ịnweta template na ọrụ nke polymerase.Onye nchịkọta akụkọ na-atụ aro na ị nwere ike ịnwale nchịkọta dị iche iche nke DSMO, dị ka site na 2% ruo 10%, ịchọta ntinye nke dabara na nnwale gị.
Ncha na-abụghị ionic
Ncha na-abụghị ionic, dị ka 0.1-1% Triton X-100, Tween 20 ma ọ bụ NP-40, na-ebelata nhazi nke abụọ DNA.Ọ bụ ezie na nke a nwere ike ịbawanye mmụba nke gene template, ọ ga-ebutekwa nsogbu nke mmụba na-abụghị nke a kapịrị ọnụ.Yabụ, mgbakwunye ndị a na-arụ ọrụ nke ọma maka mmeghachi omume PCR dị ala na-enweghị irighiri ihe, mana ọ bụghị nke ọma maka mmeghachi omume PRC na-adịghị ọcha.Uru ọzọ nke nchacha na-abụghị ionic bụ mbelata mmetọ SDS.Ọtụtụ mgbe n'oge usoro mmịpụta DNA, a ga-ebute SDS na nzọụkwụ PCR, nke na-egbochi ọrụ polymerase nke ukwuu.Ya mere, ịgbakwunye 0.5% Tween-20 ma ọ bụ Tween-40 na mmeghachi omume nwere ike ịkwụsị mmetụta ọjọọ nke SDS.
Betaine_
Betaine nwere ike melite mmụba DNA site na ibelata nhazi usoro nke abụọ yana n'ozuzu ya bụ "ihe omimi" na mgbakwunye na ngwa PCR azụmahịa.Ọ bụrụ na ịchọrọ iji betaine, ị ga-etinye betaine ma ọ bụ betaine mono-hydrate (Betaine ma ọ bụ Betaine mono-hydrate), ma ọ bụghị betaine hydrochloride (Betaine HCl), gbanwee na njedebe ikpeazụ nke 1-1.7M.Betaine nwekwara ike inye aka melite nkọwapụta n'ihi na ọ na-ewepụ ndabere ngwakọta nke ndabere nke DNA agbaze/DNA denaturation.
Ihe mgbakwunye iji belata priming na-abụghị nke akọwapụtara
Formamide
Formamide bụ ihe mgbakwunye PCR organic a na-ejikarị.Ọ nwere ike ijikọta ya na nnukwu oghere na obere oghere dị na DNA, si otú ahụ belata nkwụsi ike nke nna ukwu DNA ukwu helix abụọ ma wedata okpomọkụ nke DNA na-agbaze.Nleta nke foramide eji na nnwale PCR na-abụkarị 1%-5%.
Tetramethylammonium chloride( TMAC)
Tetramethylammonium chloride nwere ike ịbawanye nkọwa nke hybridization (ụdị hybridization) ma mee ka ogo DNA na-agbaze.Ya mere, TMAC nwere ike wepụ priming na-abụghị nke akọwapụtara ma belata nbibi nke DNA na RNA.Ọ bụrụ na ị na-ejidegenerate primersna mmeghachi omume PCR, cheta ịgbakwunye TMAC, nke a na-ejikarị na ntinye nke 15-100mM.
Ihe mgbakwunye ndị ọzọ na-emekarị
Na mgbakwunye na ụdị mgbakwunye abụọ a kpọtụrụ aha n'elu, enwere ọtụtụ ihe mgbakwunye na-emekarị na mmeghachi omume PCR, ọ bụ ezie na ha nwere ọrụ dị iche iche, ha dịkwa oke mkpa.
Magnesium ion
Magnesium ion bụ ihe dị mkpa cofactor (cofactor) nke polymerase, ya bụ, na-enweghị magnesium ion, polymerase adịghị arụ ọrụ.Otú ọ dị, nnukwu ion magnesium nwekwara ike imetụta arụmọrụ nke polymerase.Ntinye uche nke ion magnesium na mmeghachi omume PCR ọ bụla ga-adịgasị iche.Ndị ọrụ aghụghọ (dị ka EDTA ma ọ bụ citrate), ntinye nke dNTP na protein niile na-emetụta mkpokọta ion magnesium.Yabụ, ọ bụrụ na ị nwere nsogbu na nnwale PCR gị, ị nwere ike ịgbalị ịgbanwe mkpokọta ion magnesium dị iche iche, dịka ọmụmaatụ, site na 1.0 ruo 4.0mM, yana nkeji 0.5-1mM n'etiti.
Ọ dị mma ịmara na ọtụtụ okirikiri ifriizi-thaw nwere ike ibute ịta ahụhụ nke ngwọta magnesium chloride.Ya mere, ị ga-agbari ya kpamkpam tupu ojiji ọ bụla, ma gwakọta ya nke ọma tupu iji ya.
Serum albumin(Bovine albumin, BSA)
N'ime nnwale kemịkalụ molecular, albumin serum bovine bụ ihe mgbakwunye a na-ahụkarị, ọkachasị na mmachi mgbaze enzyme na nnwale PCR.Na mmeghachi omume PCR, BSA na-enye aka n'ibelata mmetọ dị ka ogige phenolic.A na-ekwukwa na ọ nwere ike ibelata adhesion nke reactants na mgbidi nke tube ule.Na mmeghachi omume PCR, na-abụkarị ntinye nke BSA agbakwunyere nwere ike iru 0.8 mg / ml.
 
Ngwaahịa ndị emetụtara:
Onye dike PCR(ya na agba)
Onye dike PCR