I-Ultra HiFidelity PCR Kit

Ukwethembeka okuphezulu, ukucaciswa okuphezulu nokusebenza kahle kwe-PC yokuqala ye-hot-start.

I-Ultra HiFidelity PCR Kit iyisiqalo esisha sokukhulisa ukuthembeka okuphezulu se-PCR esifanele ukuhlanganiswa nokutholwa okuhlobene ne-PCR. I-Ultra HiFi DNA Polymerase equkethwe kukhithi iyi-DNA polymerase esheshayo futhi enokwethenjelwa okusha eyenziwe ubuchwepheshe be-molecular evolution. Ithuthukisa ukusondelana kwe-DNA polymerase kumathempulethi, ithuthukisa isivinini sokukhulisa namandla wokunweba we-enzyme, futhi inyuse izinga lokuphumelela kwe-PCR nokukhiqiza komkhiqizo.

Ikati. Cha Ukupakisha Usayizi
4992970 1ml
4992971 5 * 1ml
4992978 5 * 5 * 1ml

 

 


Imininingwane yomkhiqizo

Isibonelo sokuhlola

Imibuzo evame ukubuzwa

Amathegi womkhiqizo

Izici

■ Kulula ukusebenza: Le khithi inikezwa njenge-2 × premix, futhi i-PCR ingenziwa ngokumane ufake izifanekiso nama-primers.
■ Ukwethembeka okuphezulu: Ukuthembeka kuphindwe izikhathi ezingama-50 kunokwe-Taq polymerase.
■ Ukucaciswa okuphezulu: Ukusebenza okuhle kakhulu kokuqalisa okushisayo ukuqinisekisa ukucaciswa komkhiqizo ..
■ Ukukhulisa ngokushesha: Ijubane lesandiso lingafinyelela ku-10-15 sec / kb.
■ Ukwanda okunamandla: Kungakhuliswa izingcezu ze-DNA ezingafika kuma-20 kb.
■ Ukusebenza okubanzi: Ikhithi iqukethe i-PCR Enhancer futhi ilungele ukukhuliswa kwama-GC aphezulu nezifanekiso eziyinkimbinkimbi.

Ukucaciswa

Uhlobo: Ukwethembeka okuphezulu kweDNA Polymerase
Isivinini sokukhulisa: 10-15 sec / kb
Usayizi wengcezu: <20kb
Ukufaka izicelo
Isizinda Sokukhishwa Kwe-DNA Kwezicubu Ezihlukahlukene Zezitshalo:
Qaphela: Isivuno se-DNA sincike ezinhlotsheni zesampula. Zonke izinto ezingenhla zivela emaqabungeni amathenda.

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


  • Langaphambilini
  • Olandelayo:

  • product_certificate04 product_certificate01 product_certificate03 product_certificate02
    ×
    Experimental Example Isiqalo esishisayo ukuqinisekisa imininingwane yomkhiqizo
    Umdwebo 1. I-Ultra HiFi inomsebenzi omuhle kakhulu wokuqalisa ukushisa ukuqinisekisa ukucaciswa kwemikhiqizo yokukhulisa. Indlela ye-beacons yamangqamuzana isetshenzisiwe (Ma et al., Anal Biochem, 2006).
    Experimental Example Ukuthembeka okuphezulu kakhulu, izikhathi ezingama-50 eziphakeme kuneTaq Polymerase
    Umdwebo 2. Ukwethembeka kwe-Ultra HiFi kuphakame izikhathi ezingama-50 kunalokho kwe-Taq polymerase ejwayelekile. Ukuthembeka kwe-polymerization kweTaq polymerase (ngaphandle komsebenzi wokulungisa) kusetshenziswa njengereferensi.
    Experimental Example Ukukhulisa okusheshayo kanye nezicucu ezinde kungakhuliswa ngokushesha
    Umdwebo 3. I-Ultra HiFi inganweba ize ifike ku-5 sec / kb ngezingcezu ezingaphansi kuka-4 kb. Ngezicucu ezinde, isikhathi sokukhulisa singanwetshwa ngokufanele. Ezingcezwini ezingaphezu kuka-15 kb, ijubane lobubanzi lingafika kumasekhondi angama-30 / kb. M: Umaka we-TIANGEN D15000
    Experimental Example Experimental Example Experimental Example Ukuqina okunamandla nokucaciswa okuphezulu, okufundeka kalula nge-GC ephezulu kanye nezicucu ezinde ezivela emithonjeni ehlukene
    Umdwebo 4. I-Ultra HiFi inokucaciswa okuphezulu ukuqinisekisa izinga lokuphumelela lokukhulisa nobungako bomkhiqizo ezinhlotsheni ezahlukahlukene zezifanekiso.
    Imiphumela ye-Ultra HiFi yokukhulisa
    B. Imiphumela yokukhulisa i-enzyme ye-Hi-Fi yoMhlinzeki K
    Imiphumela ye-enzyme yokukhulisa i-Hi-Fi yoMhlinzeki uN
    M: Umaka we-TIANGEN D15000
    Umzila 1-5. Imiphumela yokukhulisa yezifanekiso ezinobude obuhlukile: 1. 750 bp; 2. 1 kb; 3.
    2 kb; 4. 4 kb; 5. 6 kb
    Umzila 6. Umphumela wokukhulisa wethempulethi ephezulu ye-GC: 1915 bp (GC%: 70%);
    Umzila 7-11. Umphumela wokukhulisa we-2 kb templates kusuka ku-genome ehlukahlukene: 7. Igundane; 8.
    Ilayisi; 9. Ukolo; 10. Ummbila; 11. Amagciwane;
    Umzila 12-14. 8 kb long fragment amplification mphumela: 12. Ilayisi; 13. Ummbila;
    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?
    ytry
    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