Chida cha Methylation-specipc PCR (MSP)

Chida chodziwika cha PCR chodziwika ndi Methylation.

Methylation-speci fi c PCR (MSP) Kit imapangidwa makamaka kwa makasitomala omwe amaphunzira mawonekedwe a methylation a genomic DNA ndi PCR, momwe MSP DNA Polymerase ndi thermostable polymerase yosinthidwa ndi ma antibodies, ndipo 10 × MSP PCR Buffer ndi PCR buffer yokonzedweratu MSP anachita. Imagwirizana ndi TIANGEN DNA Bisulfite Conversion Kit (4992447).

Mphaka. Ayi Kukulitsa Kukula
4992759 50 preps

Mankhwala Mwatsatanetsatane

Kayendedwe

Zitsanzo Zoyesera

FAQ

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Mawonekedwe

■ Zogulitsazo zili ndi maubwino achangu, kuphweka, chidwi chachikulu, kulunjika mwamphamvu komanso kukhazikika.

Mfundo

Mtundu: MSP DNA Polymerase
Chinsinsi: <500 ng
Mapulogalamu: Ndioyenera njira ya methylation yeniyeni ya PCR (MSP) yosanthula mawonekedwe a methylation a genomic DNA

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


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    1. Methylation-Specific PCR (MSP) Kit idagwiritsidwa ntchito kukulitsa chidutswa cha 400 bp pogwiritsa ntchito bisulfite yotengera genomic DNA ngati template yokhala ndi 20 μl.

    Experimental Exampl

    2. Kukhazikitsa kayendedwe ka PCR

    Experimental Exampl

     

    Experimental Examples 1: poonekera kukonzedwa ndi katundu A;
    2: poonekera kukonzedwa ndi katundu B;
    3: TIANGEN ankachitira chitsanzo 4: NTC; M: D2000
    Experimental Examples 1-6:
    6 kubwereza;
    7: Wogulitsa Zitsanzo zokonzedwa;
    8: NTC; Bio2-F / R ndi P16- Me-F / R: Zoyambira ziwiri zoyambira.
    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?
    ytry
    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