Ngikholwa ukuthi wonke umuntu uzohlale ehlangabezana nezinkinga ezinjalo noma ezinjalo lapho enza ukusabela kwe-PCR, kodwa eziningi zazo zingahlukaniswa ngezinkinga ezimbili eziyinhloko:

Ukukhuliswa okuncane kakhulu kwesifanekiso sofuzo (ukukhulisa);
Kuningi kakhulu ukukhulisa ufuzo okungaqondisiwe.
Ukusebenzisa izithasiselo kungenye yezindlela ezivamile zokuxazulula lezi zinkinga.Ngokuvamile indima yezithasiselo inezici ezimbili:
isakhiwo sesibiliyezakhi zofuzo (isakhiwo sesibili);
Yehlisa ukusetshenziswa okungaqondile.
Namuhla, umhleli uzokwethula kafushane izithasiselo ezivamile ekuphenduleni kwe-PCR kanye nemisebenzi yazo.
Izithasiselo ezinciphisa isakhiwo sesibili
sulfoxide(DMSO)
amasampula ofuzongokuqukethwe okuphezulu kwe-GC.Nokho, i-DMSO iphinde yehlise kakhulu umsebenzi we-Taq polymerase.Ngakho-ke, wonke umuntu kufanele alinganise ukufinyeleleka kwesifanekiso kanye nomsebenzi we-polymerase.Umhleli uphakamisa ukuthi ungazama ukugxilisa okuhlukile kwe-DSMO, njengokusuka ku-2% kuye ku-10%, ukuze uthole ukugxila okufanele ukuhlolwa kwakho.
Izihlanzi ezingezona i-ionic
Izihlanzi ezingezona i-ionic, ezifana no-0.1-1% Triton X-100, Tween 20 noma NP-40, ngokuvamile zinciphisa ukwakheka kwesibili kwe-DNA.Nakuba lokhu kungakhuphula ukukhuliswa kofuzo lwesifanekiso, kuzophinde kubangele inkinga yokukhulisa okungaqondile.Ngakho-ke, lezi zithasiselo zisebenza kahle ekuphenduleni kwe-PCR enesivuno esincane ngaphandle kwemfucumfucu, kodwa hhayi kahle kakhulu ekuphenduleni okungcolile kwe-PRC.Enye inzuzo yezihlanzi ezingezona i-ionic ukunciphisa ukungcola kwe-SDS.Ngokuvamile phakathi nenqubo yokukhipha i-DNA, i-SDS izolethwa esinyathelweni se-PCR, esivimbela kakhulu umsebenzi we-polymerase.Ngakho-ke, ukwengeza u-0.5% we-Tween-20 noma i-Tween-40 ekuphenduleni kunganciphisa imiphumela emibi ye-SDS.
I-Betaine_
I-Betaine ingathuthukisa ukukhuliswa kwe-DNA ngokunciphisa ukwakheka kwesakhiwo sesibili futhi ngokuvamile iyisengezo “semfihlakalo” kumakhithi e-PCR entengiso.Uma ufuna ukusebenzisa i-betaine, kufanele ubeke i-betaine noma i-betaine mono-hydrate (i-Betaine noma i-Betaine mono-hydrate), kodwa hhayi i-betaine hydrochloride (i-Betaine HCl), ulungise ekuhlanganiseni kokugcina kwe-1-1.7M.I-Betaine ingasiza futhi ukuthuthukisa ukucaciswa ngoba iqeda ukuncika kokwakheka kwesisekelo sokuncibilika kwe-DNA/ukuguqulwa kwe-DNA.
Izithasiselo zokunciphisa ukuqala okungaqondile
I-Formamide
I-Formamide iyisengezo se-PCR esisetshenziswa kakhulu.Ingakwazi ukuhlangana ne-groove enkulu kanye ne-groove encane ku-DNA, ngaleyo ndlela yehlise ukuzinza kwe-master DNA double helix futhi yehlise izinga lokushisa elincibilikayo le-DNA.Ukugxila kwe-formamide esetshenziswa ekuhloleni kwe-PCR kuvamise ukuba ngu-1% -5%.
I-Tetramethyli-ammonium chloride( TMAC)
I-Tetramethylammonium chloride ingakhuphula ukucaciswa kwe-hybridization (ukucaciswa kwe-hybridization) futhi ikhulise izinga lokushisa elincibilikayo le-DNA.Ngakho-ke, i-TMAC ingasusa ama-priming angaqondile futhi yehlise ukubopha kabi kwe-DNA ne-RNA.Uma usebenzisaama-primers awohlokayoekuphenduleni kwe-PCR, khumbula ukwengeza i-TMAC, evame ukusetshenziswa ekuhlanganiseni okungu-15-100mM.
Ezinye Izithasiselo Ezivamile
Ngaphezu kwezigaba ezimbili zezithasiselo okukhulunywe ngazo ngenhla, kunezithasiselo eziningi ezivamile ekuphenduleni kwe-PCR, nakuba zinemisebenzi ehlukene, nazo zibaluleke kakhulu.
Magnesium ion
I-Magnesium ion iyi-cofactor (cofactor) ebaluleke kakhulu ye-polymerase, okusho ukuthi, ngaphandle kwe-magnesium ion, i-polymerase ayisebenzi.Kodwa-ke, ama-ion amaningi e-magnesium nawo angathinta ukusebenza kahle kwe-polymerase.Ukugxila kwe-magnesium ion ekuphenduleni ngakunye kwe-PCR kuzohluka.Ama-Chelating agents (afana ne-EDTA noma i-citrate), ukugcwala kwama-dNTP namaprotheni konke kuthinta ukugcwala kwama-ion e-magnesium.Ngakho-ke, uma unezinkinga ngokuhlolwa kwakho kwe-PCR, ungazama ukushintsha ukugxiliswa kwe-magnesium ion okuhlukile, isibonelo, ukusuka ku-1.0 kuye ku-4.0mM, ngesikhawu esingu-0.5–1mM phakathi.
Kuyaqapheleka ukuthi imijikelezo eminingi yokuncibilika kweqhwa ingaholela ekuhlukaniseni okugxilile kwesixazululo se-magnesium chloride.Ngakho-ke, kufanele uyihlakaze ngokuphelele ngaphambi kokusetshenziswa ngakunye, futhi uyixube kahle ngaphambi kokuyisebenzisa.
I-albumin ye-serum yenkomo(Bovine albumin, BSA)
Ezivivinyweni zekhemikhali yamangqamuzana, i-albumin ye-bovine serum iyisengezo esivame kakhulu, ikakhulukazi ekugayweni kwe-enzyme okukhawulelwe kanye nokuhlolwa kwe-PCR.Ekuphenduleni kwe-PCR, i-BSA iyasiza ekwehliseni ukungcola okufana nenhlanganisela ye-phenolic.Futhi kuthiwa kunganciphisa ukunamathela kwama-reactants odongeni lwe-tube yokuhlola.Ekuphenduleni kwe-PCR, ngokuvamile ukugxila kwe-BSA okungeziwe kungafinyelela ku-0.8 mg/ml.
 
Imikhiqizo Ehlobene:
PCR Iqhawe(nodayi)
PCR Iqhawe