Magazi Otsogolera PCR Kit

Kukulitsa mwachangu jini lomwe likufuna mwachindunji kugwiritsa ntchito magazi ngati template yopanda.

Chida ichi chimagwiritsa ntchito anti-inhibitor DNA polymerase yopanga chibadwa kuti ikwaniritse bwino majini amtundu umodzi mumtundu wamunthu. Chida cholimbitsa bwino mu chida ichi chimathandiza ma polymerase kukana mwamphamvu choletsa ma PCR inhibitors, potero amatha kukulitsa mwachindunji DNA pogwiritsa ntchito magazi ndi ma cell otsogola ngati ma tempulo. Izi ndizosavuta kuyigwiritsa ntchito ndipo sizitengera njira zovuta monga kuyeretsa kwa DNA kapena kuyeserera koyambirira.
Chikwama ichi chimaperekedwa ngati 2 × MasterMix, ndipo mayankho amatha kuchitidwa pongowonjezera template yamagazi ndi zoyambira zofananira. Itha kugwiritsidwa ntchito pamaselo otukuka a nyama monga anthu, mbewa, nkhumba, ng'ombe ndi mitundu ina, komanso mwazi watsopano kapena 4 ℃ wosungunuka, anticoagulant (EDTA, citrate, heparin), magazi oundana ndi magazi owuma zasungidwa pa makhadi amalonda a Whatman 903 ndi FTA Elute.

Mphaka. Ayi Kukulitsa Kukula
4992529 20 ×l × 100 rxn
4992530 20 ×l × 500 rxn

Mankhwala Mwatsatanetsatane

Chitsanzo Cha Zoyesera

FAQ

Zogulitsa

Mawonekedwe

■ Zosavuta komanso zachangu: Kukulitsa kwa PCR kumatha kuchitidwa mwachindunji pogwiritsa ntchito magazi ngati template, osafunikira njira zolemetsa zokonzekera ndikukonzekera DNA.
■ Kuyera kwambiri: Kudumpha kwa njira zoyeserera chithandizo komanso njira zoyeserera za DNA zitha kuthandiza kupewa kuwonongeka kwa zitsanzo.
■ Kutulutsa kwakukulu: Chizindikiritso cha PCR cha zitsanzo zazikulu zitha kuchitidwa pophatikiza zida ndi mbale za PCR za 96/384-zitsime.
■ Zapadziko lonse lapansi: Chikwama ichi chitha kukulitsa bwino zidutswa zapamwamba za GC kapena zidutswa zokhala ndi zovuta zina, ndipo kutalika kwake kungakhale mpaka 5 kb.
■ Kulimbana ndi kupanikizika kwamphamvu: Chida ichi chitha kugwiritsidwa ntchito pamitundu yosiyanasiyana ndi magazi omwe amasungidwa munjira zosiyanasiyana.

Mapulogalamu

Zogulitsa za PCR za chida ichi zili ndi "A" kumapeto kwa 3', komwe kungagwiritsidwe ntchito mwachindunji pakupanga ma vekitala a TA. Chida ichi chitha kugwiritsidwa ntchito kukulitsa tizidutswa ta genomic DNA, kusanthula kwapamwamba kwambiri kwa majini ndi kuwunika kwa genotyping (monga kuwunika kwa majini).

Zogulitsa zonse zimatha kusinthidwa kukhala ODM / OEM. Zambiri,chonde dinani Makonda Service (ODM / OEM)


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    Experimental Example Pogwiritsa ntchito anticoagulation ya anthu ngati template, majini a 4 okhala ndi mitundu yosiyanasiyana ya GC adakulitsidwa ndi Blood Direct PCR Kit. Njira yoyankhira PCR inali 20 μl, ndipo magazi a 1 μl amagwiritsidwa ntchito ngati template.
    M: TIANGEN Chikhomo II; 1: Kukula kwa zidutswa 1090 bp, GC zili 68.1%; 2: Kukula kwa zidutswa 1915 bp, GC zili 70.4%; 3: Kukula kwa zidutswa 448 bp, GC zili 74.8%; 4: Chidutswa cha kukula kwa 1527 bp, GC zili 61.5%.
    Zotsatira zoyesera: Blood Direct PCR Kit itha kukulitsa bwino zidutswa za DNA ndi zomwe zili ndi GC pamtundu wa 61.5% -74.8%, ndikuwonetsa kuti ndizotheka kukulitsa zidutswa za GC.
    Experimental Example Kugwiritsa ntchito anticoagulation ya anthu EDTA ngati template, majini 5 okhala ndi kutalika kosiyanasiyana (ActB, Prp, DN1.0, Hn2.0 ndi Hn4.0) adakulitsidwa ndi Blood Direct PCR Kit. Njira yoyankhira PCR inali 20 μl, ndipo magazi a 1 μl amagwiritsidwa ntchito ngati template.
    M: TIANGEN Chikhomo II; 1-3: 3 zitsanzo zosiyanasiyana zamagazi; NTC: kuwongolera popanda zoyambira. Zotsatira zoyesera: Blood Direct PCR Kit itha kukulitsa zidutswa zazitali kutalika ngati 4 kb, ndikuwonetsa kuti ndizotheka kukulitsa zidutswa zazitali.
    Experimental Example Pogwiritsa ntchito anticoagulation ya anthu ngati template, Blood Direct PCR Kit idagwiritsidwa ntchito pakuzindikira kwa PCR mitundu yosiyanasiyana yamagazi. Njira yoyankhira PCR inali 20 μl, ndipo magazi a 1 μl amagwiritsidwa ntchito ngati template.
    M: TIANGEN Chikhomo II; 1-9: kutsitsa magazi ndi 0.1 μl, 0.2 μl, 0.3 μl, 0.4 μl, 1 μl, 2 μl, 3 μl, 4 μl ndi 5 μl, motsatana; NTC: kuwongolera opanda template
    Zotsatira zoyesera: Blood Direct PCR Kit imalimbana mwamphamvu ndi magazi ndipo imatha kukulitsa zitsanzo zamagazi ndikutsitsa kwa 0.1-5 μl.
    Experimental Example Zitsanzo zamagazi kuchokera kwa anthu, makoswe, nkhuku ndi mitundu ina yamankhwala osiyanasiyana adagwiritsidwa ntchito ngati ma tempulo. Blood Direct PCR Kit idagwiritsidwa ntchito kukulitsa PRNP (anthu, 750 bp), Actin (rat, 200 bp), ndi β-Actin (Chicken, 1.0 kb). Njira yoyankhira PCR inali 20 μl, ndipo magazi a 1 μl amagwiritsidwa ntchito ngati template. M: TIANGEN Chikhomo II.
    Zotsatira zoyesera: Blood Direct PCR Kit itha kugwiritsidwa ntchito pamitundu ingapo, ndipo kuzindikira PCR molunjika kumatha kuchitidwa pamasampuli amwazi kuchokera ku mitundu yosiyanasiyana yamankhwala osiyanasiyana.
    Q: Palibe magulu okweza

    Chizindikiro cha A-1

    ■ Chikhazikitsochi chimakhala ndi zonyansa zomanga thupi kapena Taq inhibitors, ndi zina zambiri. —— Yembekezerani template ya DNA, chotsani zonyansa zamapuloteni kapena chotsani template ya DNA yokhala ndi zida zoyeretsera.

    ■ Kutengera kwa template sikokwanira ——Kuchulukitsa kutentha kwa chiwonetsero ndikuchulukitsa nthawi yachinyengo.

    ■ Kuwonongeka kwa ma template - -Konzani templateyo.

    Choyamba cha A-2

    ■ Zoyambira zoyipa - - Re-synthesize the primer.

    ■ Kuwonongeka koyambirira ——Gwiritsani ntchito zoyambira zazing'ono kwambiri kuti zikhale zocheperako kuti zisungidwe. Pewani kuzizira kangapo kapena kusungunuka kapena kutalika kwa 4 ° C kosungidwa.

    Kupanga koyambira koyenera (mwachitsanzo kutalika koyambirira sikokwanira, kupendekera komwe kumapangidwa pakati pazoyambira, ndi zina zambiri) -Konzani zoyambira (pewani kapangidwe kazoyambira ndi kapangidwe kake)

    A-3 Mg2+ndende

    ■ Mg2+ ndende ndiyotsika kwambiri -—Kuchulukitsa moyenera Mg2+ ndende: Konzani Mg2+ kusakanikirana ndi zochitika zingapo kuchokera 1 mM mpaka 3 mM pakadutsa 0.5 mm kuti adziwe Mg woyenera2+ ndende iliyonse ya template ndi choyambira.

    A-4 Kutentha kwa Annealing

    ■ Kutentha kokulumikiza kwakukulu kumakhudza kumangiriza koyambirira ndi template. —Chepetsani kutentha komwe kumakulitsa ndikuwonjezera vutoli ndi gradient ya 2 ° C.

    Nthawi yowonjezera-A-5

    ■ Kuonjezera nthawi yayifupi —— Wonjezerani nthawi yowonjezera.

    Funso: Chabodza

    Phenomena: Zoyipa zoyeserera zikuwonetsanso magulu omwe akutsata.

    A-1 Kuwonongeka kwa PCR

    ■ Kuwonongeka kwapakati pazinthu zomwe zikulondoleredwa kapena zopititsa patsogolo -—Mosamala kuti musapopetse sampuli yomwe ili ndi cholowa munjira yoyipa kapena kuwataya kunja kwa chubu cha centrifuge. Ma reagents kapena zida ziziyendetsedwa ndi autoclaved kuti zithetse ma nucleic acid omwe alipo, ndipo kupezeka kwa kuipitsidwa kuyenera kutsimikizika kudzera pakuyesa kosayenerera koyeserera.

    ■ Kuwonongeka kwa reagent ——Pezani mankhwala osungunula ndi kusunga pa kutentha kochepa.

    A-2 Yaikulur

    ■ Mg2+ ndende ndiyotsika kwambiri -—Kuchulukitsa moyenera Mg2+ ndende: Konzani Mg2+ kusakanikirana ndi zochitika zingapo kuchokera 1 mM mpaka 3 mM pakadutsa 0.5 mm kuti adziwe Mg woyenera2+ ndende iliyonse ya template ndi choyambira.

    ■ Mapangidwe oyambira osayenera, ndipo kutsata kwake kumayanjana ndi maphunziro aumwini osagwirizana ndi cholinga chake. ——Zipangitseni zojambula zoyambirira.

    Q: Kukulitsa kwapadera

    Phenomena: Magulu owonjezera a PCR sagwirizana ndi kukula komwe kumayembekezereka, kaya yayikulu kapena yaying'ono, kapena nthawi zina magulu amakulitsidwe ndi magulu osakondera omwe amapezeka.

    Chiyambi cha A-1

    ■ Kusazindikira koyambirira

    ——Konzaninso choyambirira.

    ■ Kutengera koyambira kumakhala kochuluka kwambiri ——Kuchulukitsa moyenera kutentha kwa chiwonetsero chazitali ndikutalikitsa nthawi yakusintha.

    A-2 Mg2+ ndende

    ■ Mg2+ ndende ndiyokwera kwambiri ——Kuchepetsa moyenera ndende ya Mg2 +: Konzani Mg2+ kusakanikirana ndi zochitika zingapo kuchokera 1 mM mpaka 3 mM pakadutsa 0.5 mm kuti adziwe Mg woyenera2+ ndende iliyonse ya template ndi choyambira.

    A-3 Thermostable polymerase

    ■ Kuchuluka kwa ma enzyme —— Chepetsani kuchuluka kwa ma enzyme moyenera pakadutsa 0.5 U.

    A-4 Kutentha kwa Annealing

    ■ Kutentha kokulumikiza ndikotsika kwambiri ——

    Zozungulira A-5 PCR

    ■ Zozungulira za PCR zochulukirapo —— Chepetsani kuchuluka kwamachitidwe a PCR.

    Q: Magulu olimba kapena opaka

    Chiyambi cha A-1——Kusalongosoka kwapafupipafupi ——Konzaninso choyambacho, sinthani malo ndi kutalika kwa zoyambira kuti zithandizire; kapena kuchita PCR yokhazikika.

    A-2 Chinsinsi DNA

    ——Tempempheroli sili loyera ——Tsukitsani template kapena tulutsani DNA yokhala ndi zida zoyeretsera.

    A-3 Mg2+ ndende

    - —Mw2+ ndende ndiyokwera kwambiri ——Chepetsani Mg2+ ndende: Konzani Mg2+ kusakanikirana ndi zochitika zingapo kuchokera 1 mM mpaka 3 mM pakadutsa 0.5 mm kuti adziwe Mg woyenera2+ ndende iliyonse ya template ndi choyambira.

    A-4 dNTP

    ——Kuchuluka kwa ma dNTP ndiokwera kwambiri —— Chepetsani kuchuluka kwa dNTP moyenera

    A-5 Kutentha kwa Annealing

    ——Kutentha kochepa kwambiri koloŵeza ——

    Zozungulira A-6

    —Zochuluka kwambiri m'zinthu —— Yambitsani kuchuluka kwa kayendedwe kake

    Q: Ndi ma template angati a DNA omwe akuyenera kuwonjezeredwa mu 50 μl PCR reaction system?
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    Q: Momwe mungakulitsire zidutswa zazitali?

    Gawo loyamba ndikusankha ma polymerase oyenera. Wokhazikika Taq polymerase sangathe kuwerengera chifukwa chosowa 3'-5 'exonuclease, ndipo kusamvana kumachepetsa kwambiri kukulitsa kwa zidutswa. Chifukwa chake, Taq polymerase wamba sangathe kukulitsa bwino zidutswa zokulirapo zopitilira 5 kb. Taq polymerase yosinthidwa mwapadera kapena kukhulupirika kwakukulu kwa polymerase kuyenera kusankhidwa kuti ikuthandizire kukulitsa zowonjezera ndikukwaniritsa zosowa zamagawo ataliatali. Kuphatikiza apo, kukulitsa kwa zidutswa zazitali kumafunikanso kusintha kofananira kwa kapangidwe koyambira, nthawi yoperekera nthawi, nthawi yowonjezera, pH ya buffer, ndi zina zambiri. Nthawi zambiri, zopangira 18-24 bp zimatha kubweretsa zokolola zabwino. Pofuna kupewa kuwonongeka kwa template, nthawi yodzitchinjiriza yomwe ili pa 94 ​​° C iyenera kuchepetsedwa kukhala 30 sec kapena kuchepera pakazungulira, ndipo nthawi yokwera kutentha mpaka 94 ° C kukweza kusanachepe 1 min. Kuphatikiza apo, kukhazikitsa kutentha kwazitali pafupifupi 68 ° C ndikupanga nthawi yowonjezera malinga ndi kuchuluka kwa 1 kb / min kumatha kutsimikizira kukulitsa kwa zidutswa zazitali.

    Q: Kodi mungakonze bwanji kukulitsa kukhulupirika kwa PCR?

    Kuchuluka kwa zolakwika za PCR kumatha kuchepetsedwa pogwiritsa ntchito ma polymerases a DNA mosadukiza. Mwa ma Taq DNA polymerases onse omwe apezeka mpaka pano, enzyme ya Pfu ili ndi zolakwika zotsika kwambiri komanso kukhulupirika kwambiri (onani tebulo lolumikizidwa). Kuphatikiza pa kusankha kwa ma enzyme, ofufuza amatha kupititsa patsogolo kusintha kwa kusintha kwa PCR pokonza momwe zinthu zikuyendera, kuphatikiza kukhathamiritsa kophatikizira, kuchuluka kwa ma polymerase osakanikirana ndikukwaniritsa kuchuluka kwa PCR.

    Lembani uthenga wanu apa ndi kutumiza kwa ife