Olona phawu lubalaseleyo lwempendulo ye-PCR sisikhundla sayo esikhulu sokukhulisa kunye novakalelo oluphezulu kakhulu.Ukuze kuphuculwe ukusebenza kwe-PCR kunye nokusebenza kakuhle, sizibophelele ekuphuculeni umthamo wokukhulisa i-PCR kunye nokuqonda kokuqonda, kodwa intloko ebuhlungu ikwinkqubo yovavanyo.Iimpawu zobuxoki zihlala zisenzeka, kwaye inani elincinci kakhulu lesampulu yongcoliseko olunqamlezayo okanye ungcoliseko lwemveliso yePCR lunokubangela iziphumo ezingeyonyani kuvavanyo.
Iintlobo ezintlanu zokungcoliseka kwemveliso ye-PCR
Zininzi izizathu zokungcoliseka kwe-PCR, ezinokuthi zohlulwe ngokwezi ndidi zilandelayo:
Ungcoliseko lwesampulu lubangelwa ikakhulu kukungcoliseka kwesikhongozeli sokuqokelela umfuziselo, okanye xa umfuziselo ubekwe, uvuza ngaphandle kwesikhongozeli ngenxa yokutywinwa okuxeliweyo, okanye umfuziselo uncamathele kumphandle wesikhongozelo, nto leyo ebangela ungcoliseko olunqamlezayo;Ungcoliseko lukhokelela kungcoliseko phakathi kwesampulu;ezinye iisampulu zemicrobial, ngakumbi iintsholongwane, zinokusasazeka nge-aerosols okanye zenze ii-aerosols, ezikhokelela ekungcolisekeni okufanayo.
Isizathu esona sizathu kukuba ngexesha lokulungiswa kwe-PCR reagents, isampuli yompu, isitya, amanzi adibeneyo aphindwe kabini kunye nezinye izisombululo zihlanjululwe yi-PCR nucleic acid template.
Kwiilabhoratri zebhayoloji yemolekyuli nakwezinye iilabhoratri ezisebenzisa iiplasmids ezicwilisiweyo njengolawulo olulungileyo, ingxaki yosulelo lweplasmid eyicloned nayo ixhaphakile.Ngenxa yokuba umxholo we-cloning plasmid kumthamo weyunithi uphezulu kakhulu, kwaye izixhobo ezingakumbi kunye nee-reagents ziyafuneka kwinkqubo yokucoca, kwaye i-plasmid kwiiseli eziphilayo kunokwenzeka ukuba ingcoliseke ngenxa yokukhula okunamandla kunye nokukwazi ukuzala kweeseli eziphilayo.
Ungcoliseko lweemveliso ezandisiweyo yeyona ngxaki ixhaphakileyo yosulelo kwiimpendulo zePCR.Ngenxa yokuba ubungakanani bekopi yemveliso ye-PCR inkulu (ngokuqhelekileyo iikopi ezili-1013/ml), engaphezulu kakhulu kunomda wenombolo yekopi yokuchongwa kwe-PCR, isixa esincinci kakhulu sokungcoliseka kwemveliso ye-PCR kunokubangela iziphumo zobuxoki.
Ungcoliseko lwe-aerosol lolona hlobo lunokwenzeka lokungcoliseka kweemveliso ze-PCR, kwaye ikwayeyona ilula ukuba ingahoywa.Yenziwa kukungqubana phakathi kolwelo kunye nomoya.Ngokuqhelekileyo, ukungcoliswa kwe-aerosol kunokubunjwa xa i-lid ivuliwe, xa isampuli inqwenela, okanye nangona ityhubhu yokusabela ishukunyiswa ngamandla.Ngokwezibalo, i-particle ye-aerosol ingaba neekopi ze-48,000, ngoko ke ukungcola okubangelwa yingxaki efanelwe ingqalelo ekhethekileyo.
Ngokukodwa, iilabhoratri zovavanyo zihlala zisebenzisa ipere efanayo yeeprimers ukuvavanya uhlobo oluthile lwemfuza.Ngokuhamba kwexesha, inani elikhulu lokungcoliseka kwemveliso ye-PCR liya kwenzeka kwindawo yebhubhoratri.Emva kokuba ungcoliseko olunjalo lwenzekile, kunzima ukuluphelisa ngexesha elifutshane.
Kwiintlobo ezintathu zokuqala zongcoliseko, sinokusebenzisa iindlela ezisebenzayo zokuphepha, kodwa ungcoliseko olubangelwa iimveliso ze-PCR kunzima ukukuthintela, ngakumbi ekwakhiweni kwee-laboratories ze-PCR ezingekho-standard.Kwinkqubo ye-PCR, xa i-tip ye-pipette ifunxa kwaye ivuthela i-liquid, kwaye i-tube ye-PCR ikhava ivuliwe, i-aerosol iya kwenziwa.Iimolekyuli ze-DNA ezithwalwa yi-aerosol (i-aerosol enye inokuthwala amashumi amawaka e-DNA) kunzima ukuyiphelisa kuba idada emoyeni.Nje ukuba umjikelo olandelayo wovavanyo lwe-PCR uqaliswe, iziphumo ezingezizo ezingeyonyani ziyakwenzeka.
Njengoko kubonisiwe kulo mzobo ungezantsi, ulawulo olubi lukwakhulise ibhanti yomdla ohambelana nayo:
Inxalenye yokuqala yalo mba wongcoliseko lwePCR kunye nothintelo yaziswa apha.Inqaku elilandelayo liza kukuzisela inxalenye yesibini "Ukuthintela ukungcoliswa kwemveliso ye-PCR", ke hlala ubukele!
Ixesha lokuthumela: Jul-25-2017
