2 × I-Pfu PCR Mix

Isifanekiso se-A-1

■ Ithempulethi iqukethe ukungcola kwamaprotheni noma ama-Taq inhibitors, njll. - —Purify DNA template, susa ukungcola kwamaprotheni noma ukhiphe i-DNA template ngamakhithi okuzihlanza.

■ Ukwehlukaniswa kwethempulethi akuphelele ——Kwandisa ngokufanele ukushisa kwama-denaturation futhi kwandise isikhathi se-denaturation.

■ Ukuwohloka kwesifanekiso ——Lungisa kabusha ithempulethi.

I-A-2 Primer

■ Izinga eliphansi lama-primers ——Re-synthesize the primer.

■ Ukuwohloka kwe-Primer ——Faka ama-primer primer aphezulu abe yivolumu encane yokulondolozwa. Gwema ukubanda okuningi nokuncibilikisa noma ukugcina isikhathi eside u-4 ° C.

■ Idizayini engalungile yama-primers (isb. Ubude be-primer abwenele, i-dimer eyakhiwe phakathi kwama-primers, njll.) -Redesign primers (gwema ukwakheka kwe-primer dimer nesakhiwo sesibili)

I-A-3 Mg²⁺ukuhlushwa

■ Mg²⁺ ukuminyana kuphansi kakhulu —— Khulisa ngokufanele uMg²⁺ ukuhlushwa: Lungiselela iMg²⁺ ukugxila ngochungechunge lokuphendula kusuka ku-1 mM kuye ku-3 mM ngesikhawu esingu-0.5 mM ukuthola iMg efanelekile²⁺ ukugxila kuthempulethi ngayinye nakuqala.

Ukushisa kwe-A-4 Annealing

The Ukushisa okuphezulu kokunciphiswa komthelela kuthinta ukubopha kwe-primer ne-template. - - Nciphisa ukushisa okunciphisayo futhi wenze ngcono isimo nge-gradient ka-2 ° C.

Isikhathi se-A-5 Sesandiso

■ Isikhathi esifushane sokunweba — - Khulisa isikhathi sokunweba.

I-Phenomena: Amasampula amabi nawo akhombisa amabhendi wokulandelana okuqondiwe.

Ukungcola kwe-A-1 kwe-PCR

■ Ukungcola kwesiphambeko sokulandelana okuqondiwe noma imikhiqizo yokukhulisa amandla ——Nakekela ukuthi ungafaki isampuli ngesampula equkethe ukulandelana kwethagethi kusampula engeyinhle noma uchithe iphume kushubhu le-centrifuge. Ama-reagents noma okokusebenza kufanele kwenziwe ngokuzenzakalela ukuqeda ama-nucleic acid akhona, futhi ubukhona bokungcola kufanele kunqunywe ngokuhlolwa kokulawulwa okungalungile.

■ Ukungcola okuyisenzo ——Faka ama-reagents bese uwagcina lapho kushisa okuncane.

I-A-2 Primer

■ Mg²⁺ ukuminyana kuphansi kakhulu —— Khulisa ngokufanele uMg²⁺ ukuhlushwa: Lungiselela iMg²⁺ ukugxila ngochungechunge lokuphendula kusuka ku-1 mM kuye ku-3 mM ngesikhawu esingu-0.5 mM ukuthola iMg efanelekile²⁺ ukugxila kuthempulethi ngayinye nakuqala.

■ Idizayini yokuqala engafanele, nokulandelana kwempokophelo kune-homology ngokulandelana okungeyona eyenhloso. ——Kwakha kabusha ama-primers.

I-Phenomena: Amabhendi wokukhulisa we-PCR awahambelani nosayizi olindelekile, kungaba mikhulu noma mincane, noma kwesinye isikhathi womabili amabhendi okukhulisa kanye namaqembu wokukhulisa angaqondile.

I-A-1 Primer

■ Ukucaciswa okungafanelekile kokuqala

——Kwakha kabusha isiqalo.

■ Ukuhlungwa kwe-primer kuphakeme kakhulu — -Kwandisa ngokufanele ukushisa kwe-denaturation futhi kwandise isikhathi se-denaturation.

I-A-2 Mg²⁺ ukuhlushwa

■ UMg²⁺ ukuminyana kuphakeme kakhulu —— Nciphisa kahle ukuhlushwa kwe-Mg2 +: Lungiselela iMg²⁺ ukugxila ngochungechunge lokuphendula kusuka ku-1 mM kuye ku-3 mM ngesikhawu esingu-0.5 mM ukuthola iMg efanelekile²⁺ ukugxila kuthempulethi ngayinye nakuqala.

I-A-3 Thermostable polymerase

■ Inani le-enzyme eyeqile - - Nciphisa inani le-enzyme ngokufanele ngezikhathi ezithile zika-0.5 U.

Ukushisa kwe-A-4 Annealing

■ Izinga lokushisa elinciphisayo liphansi kakhulu ——Kwandisa ngokufanele ithempelesha yokuncisha noma ukusebenzisa indlela yokuncisha izigaba ezimbili

Imijikelezo ye-A-5 PCR

■ Imijikelezo eminingi kakhulu ye-PCR —— Nciphisa inani lemijikelezo ye-PCR.

I-A-1 Primer—— Okucacile okungekuhle—— Yakha kabusha i-primer, shintsha ukuma nobude be-primer ukuthuthukisa ukucaciswa kwayo; noma wenze i-PCR ehlanganisiwe.

I-A-2 Template DNA

——Isifanekiso asihlanzekile —Hlanza ithempulethi noma ukhiphe i-DNA enezikhwama zokuhlanzwa.

I-A-3 Mg²⁺ ukuhlushwa

- —Mg²⁺ ukugxilisa ingqondo kuphakeme kakhulu ——Ukunciphisa kahle uMg²⁺ ukuhlushwa: Lungiselela iMg²⁺ ukugxila ngochungechunge lokuphendula kusuka ku-1 mM kuye ku-3 mM ngesikhawu esingu-0.5 mM ukuthola iMg efanelekile²⁺ ukugxila kuthempulethi ngayinye nakuqala.

I-A-4 dNTP

—— Ukuhlungwa kwama-dNTP kuphakeme kakhulu —— Nciphisa ukuminyana kwe-dNTP ngokufanele

Ukushisa kwe-A-5 Annealing

——Ukushisa okuncishisayo okuphansi impela —— Kukhuphula kahle ukushisa okuncishisayo

Imijikelezo engu-A-6

—Imijikelezo eminingi kakhulu —— Yandisa inani lomjikelezo

Isinyathelo sokuqala ukukhetha i-polymerase efanelekile. I-Taq polymerase ejwayelekile ayikwazi ukufundwa ngenxa yokuntuleka komsebenzi we-3'-5 'wokuxolelwa, futhi ukungafani kahle kuzonciphisa kakhulu ukusebenza kwezingcezu. Ngakho-ke, i-Taq polymerase ejwayelekile ayikwazi ukukhulisa ngempumelelo izingcezu ezihlosiwe ezinkulu kune-5 kb. I-Taq polymerase enokuguqulwa okukhethekile noma okunye ukuthembeka okuphezulu kwe-polymerase kufanele kukhethwe ukuthuthukisa ukusebenza kahle kokunwetshwa nokuhlangabezana nezidingo zokukhulisa isiqeshana eside. Ngaphezu kwalokho, ukukhuliswa kwezingcezu ezinde kudinga nokulungiswa okuhambisanayo komklamo wokuqala, isikhathi sokudlulisa isikhathi, isikhathi eseluliwe, i-pH yesikhondlakhondla, njll. Ngokuvamile, ama-primers ane-18-24 bp angaholela esivunweni esingcono. Ukuvikela ukulimala kwethempulethi, isikhathi sokudonswa kwenani elingu-94 ° C kufanele sehliswe lifike kumasekhondi angama-30 noma ngaphansi ngomjikelezo ngamunye, nesikhathi sokukhuphuka kwamazinga okushisa siye ku-94 ° C ngaphambi kokukhulisa amandla kufanele kube ngaphansi kweminithi elilodwa. Ngaphezu kwalokho, ukusetha izinga lokushisa lokunwebeka cishe ku-68 ° C nokuklama isikhathi sesandiso ngokuya ngesilinganiso se-1 kb / min kungaqinisekisa ukukhuliswa okusebenzayo kwezingcezu ezinde.

Izinga lephutha lokukhulisa i-PCR lingehliswa ngokusebenzisa ama-polymerase e-DNA ahlukahlukene ngokuthembeka okuphezulu. Kuwo wonke ama-polymerase eTaq DNA atholakele kuze kube manje, i-Pfu enzyme inezinga lephutha eliphansi kakhulu nokuthembeka okuphezulu (bheka ithebula elihlanganisiwe). Ngaphezu kokukhethwa kwe-enzyme, abacwaningi bangaqhubeka banciphise izinga lokuguqulwa kwe-PCR ngokwenza ngcono izimo zokuphendula, kufaka phakathi ukwakheka kwe-buffer, ukuminyana kwe-polymerase esetshenziswayo nokwandisa inombolo yomjikelezo we-PCR.