Toto Direct PCR Pusa

Faʻateleina le faʻatupulaia o le fua faʻatulagaina tuʻusaʻo faʻaaogaina le toto o se faʻataʻitaʻiga e aunoa ma se toʻesea mai.

Lenei pusa faʻaaogaina le genetically engineered anti-inhibitor DNA polymerase ia lelei faʻalauteleina tasi-kopi kenera i le genome tagata. O le lelei faʻalelei buffer system i lenei pusa fesoasoani le polymerase matua teteʻe i le taofiofia o PCR taofia, o lea e mafai ai ona faʻateleina DNA faʻateleina le faʻaaogaina o le toto ma sela faʻavasegaina pei o faʻataʻitaʻiga. Lenei oloa e faigofie ona faʻagaioia ma e le manaʻomia laʻasaga faigata e pei o DNA faʻamamaina poʻo faʻataʻitaʻiga pretreatment.
Lenei pusa ua faʻaavanoaina o le 2 × MasterMix, ma o le tali atu e mafai ona faia i le na o le faʻaopopoina o le toto faʻataʻitaʻiga ma tutusa mauaina muamua faʻamuamua. E mafai ona faʻaogaina i luga o sela faʻapipiʻiina o meaola mamalia e pei o tagata, isumu, puaʻa, povi ma isi ituaiga, faʻapea foi ma le fou poʻo le 4 ℃ cryopreserve toto atoa, anticoagulant (EDTA, citrate, heparin), liu faʻasolo toto ma mugala toto teu i luga Whatman 903 ma FTA Elute pisinisi pisinisi.

Pusi leai Tele teuina
4992529 20 µl × 100 rxn
4992530 20 µl × 500 rxn

Oloa Faʻamatalaga

Faʻataʻitaʻiga Faʻataʻitaʻiga

FAQ

Pine o oloa

Features

■ Faigofie ma vave: PCR amplification mafai ona faʻatinoina faʻaaoga faʻaaoga le toto e pei o template, aunoa ma le manaʻoga mo le faigata laʻasaga o le faʻataʻitaʻiga sauniuniga ma DNA toʻesea.
■ Maualuga mama: O le faʻamisi o faʻataʻitaʻi muamua togafitiga ma DNA aveʻese laʻasaga mafai ona fesoasoani e aloese ai mai le satauro-faʻaleagaina o faʻataʻitaʻiga.
■ Maualuga maualuga: O le PCR faʻailoaina mo tele-fua faʻataʻitaʻiga mafai ona faia e ala i le tuʻufaʻatasia o le ato ma 96/384-lelei PCR ipu.
■ Malosi lautele: O lenei pusa e mafai ona faʻateleina maualuga GC vaega poʻo vaega ma laʻititi lona lua fausaga, ma le faʻateleina umi mafai ona oʻo atu i le 5 kb.
■ Tetee malosi ile teteʻe: E mafai ona faʻaoga lenei pusa mo ituaiga eseese ma faʻataʻitaʻiga ole toto na faʻasaoina i auala eseʻese.

Talosaga

O oloa a le PCR o lenei ato o loʻo iai le "A" i le 3'-end, lea e mafai ona faʻaaoga saʻo mo le TA vector cloning. Lenei pusa e mafai ona faʻaaogaina mo le faʻateleina o genomic DNA fragments, maualuga-throughput gaʻo auʻiliʻiliga ma genotyping (e pei o le mauaina o gen) suʻesuʻega.

Uma oloa mafai ona faʻapitoa mo ODM / OEM. Mo auiliiliga,faʻamolemole kiliki Customized Service (ODM / OEM)


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    Experimental Example Faʻaogaina o le tagata EDTA anticoagulation o se faʻataʻitaʻiga, 4 genes ma eseʻese GC mea na faʻateleina e le Blood Direct PCR Kit. O le PCR tali faʻatonuga na 20 μl, ma le 1 μl toto na faʻaaogaina e fai ma faʻataʻitaʻiga.
    M: TIANGEN Faʻailoga II; 1: Fuafua tele 1090 bp, GC anotusi 68.1%; 2: Fuafua tele 1915 bp, GC anotusi 70,4%; 3: Fuafua tele 448 bp, GC anotusi 74.8%; 4: Fuafua tele 1527 bp, GC anotusi 61.5%.
    Faʻataʻitaʻiga iʻuga: Toto Direct PCR Pusa mafai lelei faʻalauteleina DNA vaega ma le GC anotusi i le tulaga o 61.5% -74.8%, fautua mai e mafai ona faʻateleina maualuga-GC fasi.
    Experimental Example Faʻaogaina o le tagata EDTA anticoagulation o se faʻataʻitaʻiga, 5 genes ma eseese uumi (ActB, Prp, DN1.0, Hn2.0 ma Hn4.0) na faʻateleina e le Blood Direct PCR Kit. O le PCR tali faʻatonuga na 20 μl, ma le 1 μl toto na faʻaaogaina e fai ma faʻataʻitaʻiga.
    M: TIANGEN Faʻailoga II; 1-3: 3 faʻataʻitaʻiga toto; NTC: faʻatonutonu e aunoa ma le muamua. Faʻataʻitaʻiga iʻuga: Toto Direct PCR Pusa mafai faʻalauteleina vaega ma le umi pe a o le 4 kb, fautua mai e mafai ona faʻalauteleina umi fasi.
    Experimental Example Faʻaogaina o le tagata EDTA anticoagulation o se faʻataʻitaʻiga, Toto Direct PCR Pusa na faʻaaogaina mo PCR mauaina o toto eseese faʻataʻitaʻiga. O le PCR tali faʻatonuga na 20 μl, ma le 1 μl toto na faʻaaogaina e fai ma faʻataʻitaʻiga.
    M: TIANGEN Faʻailoga II; 1-9: o le utaina aofaʻi o le toto o 0.1 μl, 0.2 μl, 0.3 μl, 0.4 μl, 1 μl, 2 μl, 3 μl, 4 μl ma 5 μl, i le faasologa; NTC: faʻatonutonu ma leai se faʻataʻitaʻiga
    Faʻataʻitaʻiga iʻuga: Toto Direct PCR Pusa o loʻo teteʻe malosi i le toto ma mafai faʻalauteleina toto faʻataʻitaʻiga ma le utaina tulaga o 0.1-5 μl.
    Experimental Example Toto faʻataʻitaʻiga mai le tagata, isumu, moa ma isi ituaiga ma eseʻese togafitiga na faʻaaogaina e fai ma faʻataʻitaʻiga. Toto Direct PCR Kit na faʻaaoga e faʻatele ai le PRNP (tagata, 750 bp), Actin (rat, 200 bp), ma le β-Actin (Moa, 1.0 kb). O le PCR tali faʻatonuga na 20 μl, ma le 1 μl toto na faʻaaogaina e fai ma faʻataʻitaʻiga. M: TIANGEN Faʻailoga II.
    Faʻataʻitaʻiga iʻuga: Toto Direct PCR Pusa mafai ona faʻaaogaina luga o le tele o ituaiga o faʻataʻitaʻiga, ma le faʻatonuga tuusaʻo PCR mafai ona faia luga toto faʻataʻitaʻiga mai le tele o ituaiga ma eseʻese togafitiga.
    Q: Leai ni faʻamau faʻateleina

    A-1 Faʻataʻitaʻiga

    ■ O le faʻataʻitaʻiga o loʻo i ai ni polotini polotito poʻo Taq inhibitors, ma isi.

    ■ E le o maeʻa le faʻavaeina o faʻataʻitaʻiga - - Faʻalelei talafeagai le vevela o le sauniga ma faʻafualoa le taimi o le faʻasologa.

    ■ Faʻalumaina o le faʻataʻitaʻiga —— Toe sauni le faʻataʻitaʻiga.

    A-2 Faʻatomuaga

    ■ Le lelei le lelei o faʻamuamua ——Re toe faʻafouina le amataga.

    ■ Faʻaleagaina muamua --— Faʻavae muamua le maualuga o le faʻasusu i tamaʻi tusi mo le faʻasaoina. Aloese mai le tele o le malulu ma le liusuavai poʻo le umi taimi 4 ° C cryopreserve.

    ■ Le fetaui lelei le faʻavaeina o primers (eg primer length e le lava, dimer fausia i le va o primers, ma isi) -Redesign primers (aloese mai le fausiaina o primer dimer ma tulaga lua fausaga)

    A-3 Mg2+taulaʻi

    ■ Mg2+ ua maualalo tele le faʻatatau --— Faʻalelei lelei Mg2+ faʻamalosia: Faʻalelei le Mg2+ faʻamalosia e se faʻasologa o tali mai 1 mM i le 3 mM ma le va o le 0.5 mM e fuafua ai le sili lelei Mg2+ faʻasologa mo taʻitasi template ma primer.

    A-4 Annealing vevela

    ■ O le maualuga annealing vevela aafia ai le fusia o primer ma template. —— Faʻaitiitia le vevela faʻapipiʻi ma faʻalelei le tulaga i le gradient o le 2 ° C.

    A-5 Faʻasologa o taimi

    ■ Taimi faʻaopoopo puupuu —— Faʻateleina taimi faʻaopopo.

    Q: Sese mea lelei

    Phenomena: O faʻataʻitaʻiga le lelei e faʻaalia ai foʻi faʻasologa o faʻasologa.

    A-1 Faʻaleagaina o PCR

    ■ Faʻalavaveina faʻaleagaina o faʻasologa faʻasologa poʻo le faʻateleina o oloa --— Faʻaeteete aua neʻi pipuina le faʻataʻitaʻiga o loʻo i ai le faʻasologa faʻatulagaina i le le lelei faʻataʻitaʻiga pe faʻamaligiina i fafo mai le centrifuge tube. O reagents poʻo mea faigaluega e tatau ona autoclaved e aveʻese ai nei nucleic acid, ma le i ai o faʻaleagaina tatau ona fuafuaina e ala i le le lelei faʻataʻitaʻiga faʻataʻitaʻiga.

    ■ Faʻaleagaina faʻaleagaina ——Faʻamaʻaina meaʻai ma faleoloa i le maualalo o le vevela.

    A-2 Palemiar

    ■ Mg2+ ua maualalo tele le faʻatatau --— Faʻalelei lelei Mg2+ faʻamalosia: Faʻalelei le Mg2+ faʻamalosia e se faʻasologa o tali mai 1 mM i le 3 mM ma le va o le 0.5 mM e fuafua ai le sili lelei Mg2+ faʻasologa mo taʻitasi template ma primer.

    ■ Le fetaui lelei le ata muamua, ma le faʻasologa faʻatulagaina ei ai homology ma le lē faʻatulagaina faʻasologa. ——Re-design primers.

    Q: Le faʻapitoa faʻalauteleina

    Phenomena: O faʻamau faʻalauteleina PCR e le ogatusa ma le tele fuafuaina, a le o le tele poʻo le laʻititi, poʻo nisi taimi uma faʻapitoa faʻalauteleina fusi ma le faʻapitoa faʻalauteleina fusi tupu.

    A-1 Faʻatomuaga

    ■ Lelei faʻatulagaina primer

    ——Re-design primer.

    ■ O le faʻamuamua primer e maualuga tele —— Faʻalelei lelei le denaturation vevela ma faʻafualoa taimi denaturation.

    A-2 Mg2+ taulaʻi

    ■ O le Mg2+ ua maualuga tele le faʻatonuina - - Faʻaitiitia lelei le faʻateteleina o le Mg2 +: Faʻaaoga lelei le Mg2+ faʻamalosia e se faʻasologa o tali mai 1 mM i le 3 mM ma le va o le 0.5 mM e fuafua ai le sili lelei Mg2+ faʻasologa mo taʻitasi template ma primer.

    A-3 Thermalable polymerase

    ■ Ova aofaʻi o le enzyme —— Faʻaitiitia talafeagai le aofaʻi o le enzyme i vaitaimi o le 0.5 U

    A-4 Annealing vevela

    ■ O le vevela faʻapipiʻi e maualalo tele —— Faʻalelei talafeagai le faʻapipiʻiina o le vevela pe faʻaogaina le lua-tulaga faʻamamaina auala

    A-5 PCR taʻamilosaga

    ■ Ova tele taʻamilosaga PCR —— Faʻaitiitia le aofaʻi o taʻavale PCR.

    Q: Patchy pe faʻapipiʻi fusi

    A-1 Faʻatomuaga——E le lelei le faʻamatalaina --- Le toe mamanuina o le tusi muamua, suia le tulaga ma le umi o le tusi muamua e faʻaleleia atili ai lona faʻapitoa; pe faʻataʻitaʻi PCR faʻamoe.

    A-2 Faʻataʻitaʻiga DNA

    ——E le mamā le faʻataʻitaʻiga --— Faʻamama le ata pe aveese DNA ma ato faʻamama.

    A-3 Mg2+ taulaʻi

    ——Mai2+ ua maualuga tele le faʻatonuina - - Faʻaitiitia tatau le Mg2+ faʻamalosia: Faʻalelei le Mg2+ faʻamalosia e se faʻasologa o tali mai 1 mM i le 3 mM ma le va o le 0.5 mM e fuafua ai le sili lelei Mg2+ faʻasologa mo taʻitasi template ma primer.

    A-4 dNTP

    —— O le faʻatulagaina o dNTPs e maualuga tele —— Faʻaitiitia talafeagai o le dNTP

    A-5 Annealing vevela

    ——Talu maualalo tele le vevela faʻapipiʻi —— Faʻatuputeleina le vevela faʻapipiʻi

    A-6 Taamilosaga

    —— Matua tele taʻamilosaga —— Faʻamamafa le numera ole taʻamilosaga

    Q: E fia faʻailoga DNA e tatau ona faʻaopopoina i le 50 μl PCR tali atu faʻasologa?
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    F: Faʻafefea ona faʻalautele umi fasi?

    O le laʻasaga muamua o le filifili lea o le polymerase talafeagai. Ole Taq polymerase masani e le mafai ona faʻamaonia ona o le le lava o le 3'-5 'exonuclease gaoioiga, ma le le fetaui lelei o le a matua faʻaititia ai le faʻalauteleina o le aoga o vaega. O le mea lea, o le Taq polymerase masani e le mafai ona faʻamalosia lelei vaega faʻatulagaina lapoʻa nai lo le 5 kb. Taq polymerase ma faʻapitoa suiga poʻo isi maualuga aiai ai polymerase tatau ona filifilia e faʻaleleia atili faʻalautelega lelei ma faʻamalieina ai manaʻoga o le umi fragment amplification. I se faʻaopopoga, o le faʻalauteleina o uumi vaega e manaʻomia foʻi fetuʻunaʻiga o uluaʻi mamanu, denaturation taimi, faʻaopoopoga taimi, buffer pH, ma isi masani, primers ma 18-24 bp mafai ona taʻitaʻia ai i sili lelei fua. Ina ia mafai ona puipuia le faʻaleagaina o le faʻavasega, o le taimi o le tino i le 94 ° C e tatau ona faʻaititia i le 30 sekone pe itiiti ifo i le taʻamilosaga, ma le taimi e faʻatupu ai le vevela i le 94 ° C ao le i faʻateleina le tatau ona laʻititi ifo i le 1 min. E le gata i lea, o le faʻatulagaina o le faʻalauteleina o le vevela pe a ma le 68 ° C ma le fuafuaina o le faʻaopoopoga taimi e tusa ai ma le fua faatatau o le 1 kb / min mafai ona mautinoa le faʻalauteleina aoga o umi fasi.

    Q: Faʻafefea ona faʻaleleia le faʻateleina o le faʻamaoni o le PCR?

    O le sese fua o le PCR faʻateleina mafai ona faʻaititia i le faʻaaogaina o DNA eseese polymerases ma maualuga faʻamaoni. Faʻatasi ai ma Taq DNA polymerases o loʻo maua i le taimi nei, o le Pfu enzyme o loʻo i ai le tau maualalo maualalo ma le maualuga o le faʻamaoni. I le faʻaopopoga i le filifiliga o le enzyme, e mafai e tagata suʻesuʻe ona faʻaititia le suiga o le PCR i le faʻamautinoaina o tulaga o tali atu, e aofia ai le faʻamautinoaina o le faʻapipiʻiina o le buffer, o le totoʻa o le polymerase ma le faʻamautinoaina o le numera o le taʻamilosaga a le PCR.

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