I-2 × Taq Plus PCR Mix

I-Ultra-msulwa, ukusebenza kahle okuphezulu nokuthembeka okuphezulu kweTaq DNA polymerase.

ITaq Plus DNA Polymerase iyinhlanganisela yeTaq nePfu DNA polymerase. Inomsebenzi we-5'-3 'we-exonuclease nomsebenzi we-3'-5' wokukhululwa, enezici zokukhulisa okuphezulu kanye nezinga eliphansi lokungafani. Uma kuqhathaniswa neTaq DNA polymerase, iTaq Plus DNA polymerase inezinzuzo zokukhulisa ubude bokukhulisa (izifanekiso ezilula zingakhuliswa ngempumelelo kuze kufike ku-20kb futhi izifanekiso eziyinkimbinkimbi zingafika ku-10 kb), ukuthembeka okuhle, njll. Uma kuqhathaniswa nePfu DNA polymerase, it inezinzuzo zejubane lokukhulisa ngokushesha nokusebenza kahle kokuphendula okuphezulu.

Ikati. Cha Ukupakisha Usayizi
4992791 1ml
4992792 5 * 1ml
4992793 5 × 1 ml

Imininingwane yomkhiqizo

Isibonelo sokuhlola

Imibuzo evame ukubuzwa

Amathegi womkhiqizo

Incazelo Yomsebenzi

Umsebenzi we-1 Unit (U) Taq Plus DNA Polymerase uchazwa njengenani le-enzyme elidingekayo ukufaka i-10 nmol deoxynucleotide ezintweni ezingenayo i-acid ku-74 ° C kungakapheli imizuzu engama-30 kusetshenziswa isidoda se-salmon DNA njengesifanekiso / kuqala.

Ikhwalithi yokulawula

Ukuhlanzeka ngokutholwa kwe-SDS-PAGE kungaphezu kwama-99%; Awukho umsebenzi we-nuclease wangaphandle otholakele; Uhlobo lwekhophi elilodwa ku-genome yomuntu lungakhuliswa ngempumelelo; Akukho ukushintsha komsebenzi okuphawulekayo lapho kugcinwa ekamelweni lokushisa isonto elilodwa.

Main Technical Amapharamitha

I-Taq Plus DNA Polymerase inomsebenzi wokukhululwa ngaphandle kuka-5'-3 'nomsebenzi we-3-5 Imikhiqizo ye-PCR ingahlanganiswa ngqo ku-vector ye-TA. Uma ukusebenza kahle kwe-cloning kudinga ukwenziwa ngcono, kunconywa ukuthi kuhlanzwe imikhiqizo ye-PCR kuqala futhi kwenziwe i-A-tailing ngaphambi kokuhlangana ku-vector ye-TA.

I-One-tube Taq Plus PCR Mix (Isitifiketi Sikazwelonke Semikhiqizo Yezobuchwepheshe)

■ I-Taq Plus PCR Mix ithuthukise ukucaciswa nokuzwela kokuphendula kwe-PCR futhi ingakhulisa izifanekiso eziyinkimbinkimbi ngokuqukethwe okuphezulu kwe-GC, ukwakheka kwesibili nokunye okunjalo. Amakhophi ama-2 aphansi wethempulethi ekhonjiwe angakhuliswa, aqinisekise imiphumela yokulinga enembe kakhulu.

■ Ifomula eliyingqayizivele leTaq Plus PCR Mix lenza lonke uhlelo lokuphendula luzinze kakhulu, futhi umsebenzi ngeke uthinteke ukubanda okubandayo okuphindaphindiwe noma ukugcinwa kwesikhathi eside ngo-4 ° C.

■ Isixazululo sokuxuba se-PCR esizinzile nesisebenza kahle singenza ukusebenza kusheshe futhi kube lula, kunciphise kakhulu umfutho wabasebenzi nephutha lesampula. Isithuthukisi se-PCR esisebenza kahle kanye ne-optimizer nakho kufakiwe ekuxubeni, okunciphisa izidingo ezimweni ze-PCR.

■ Lo mkhiqizo unezinhlelo zombili eziqukethe udayi nezingenawo udayi. Imikhiqizo ye-PCR Mix equkethe udayi ingaqokelwa ngqo i-electrophoresed ngemuva kwe-PCR, ngaphandle kokungeza isampula sesikhashana.

Izicelo

Ijwayele ukusetshenziselwa ukukhulisa izifanekiso ezinokwethembeka okuphezulu nesakhiwo esiyinkimbinkimbi, njengokuqukethwe okuphezulu kwe-GC nesakhiwo sesibili. Ezimweni eziningi, ingathatha isikhundla seTaq DNA polymerase.

Yonke imikhiqizo ingenziwa ngezifiso i-ODM / OEM. Ngemininingwane,sicela uchofoze i-Customized Service (ODM / OEM)


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    Experimental Example Experimental Example Sebenzisa i-genomic DNA njengethempulethi ukukhulisa ucezu lwe-1 kb.
    Ngemuva kokuphendula kwe-PCR, thatha u-5 μl ukuthola ukutholwa kwe-electrophoresis.
    Q: Awekho amabhendi wokukhulisa

    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.

    Umbuzo: Ukuthi kunamanga

    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.

    Q: Ukukhulisa okungacacisiwe

    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.

    Q: Ama-Patchy noma ama-smear bands

    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

    Q: Mangaki ithempulethi ye-DNA okufanele ingezwe ku-50 μl system reaction reaction?
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    Q: Ungazikhulisa kanjani izingcezu ezinde?

    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.

    Q: Ungakuthuthukisa kanjani ukwethembeka kokukhulisa kwe-PCR?

    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.

    Bhala umyalezo wakho lapha bese uwuthumela kithi