Monga watsopano mu labotale, si ntchito yabwino kuyang'ana zomera zabwino kuchokera ku gulu la zomera zomwe zimasintha.Choyamba, DNA iyenera kutengedwa kuchokera ku zitsanzo zambiri imodzi ndi imodzi, kenako majini akunja adzazindikiridwa ndi PCR.Komabe, zotsatira zake nthawi zambiri zimakhala zopanda kanthu komanso zokhala ndi zinthu zingapo nthawi zina, koma ndizosatheka kudziwa ngati pali kuphonya kapena kuzindikira zabodza..Kodi n'kosathandiza kukumana ndi zoyeserera zotere ndi zotsatira zake?Osadandaula, m'bale amakuphunzitsani momwe mungadziwire mbewu zomwe zili ndi kachilomboka mosavuta komanso molondola.

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Zoyambira zozindikirira mapangidwe

Tsimikizirani jini yokhazikika komanso jini yakunja kuti izindikiridwe molingana ndi chitsanzo chomwe chiyezedwe, ndikusankha woyimira 100-500bp motsatizana mu jini yopangira zoyambira.Zoyambira zabwino zimatha kuwonetsetsa kulondola kwa zotsatira zodziwikiratu ndikufupikitsa nthawi yodziwikiratu (onani zowonjezera pazowunikira zomwe zimagwiritsidwa ntchito nthawi zambiri).

Zindikirani: Zoyambira zomwe zangopangidwa kumenezi zimayenera kukhathamiritsa momwe zimachitikira ndikuwonetsetsa kulondola, kulondola komanso malire odziwikiratu asanazindikire kwakukulu.

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Pangani protocol yoyesera

Kuwongolera kwabwino: Gwiritsani ntchito DNA yoyeretsedwa yomwe ili ndi chidutswa chandamale ngati template kuti muwone ngati PCR reaction system ndi mikhalidwe ndiyabwinobwino.

Kuwongolera kolakwika/kusowekapo kanthu: Gwiritsani ntchito template ya DNA kapena ddH2O yomwe ilibe chidutswa chandamale ngati template kuti muwone ngati pali gwero la kuipitsidwa mu PCR system.

Ulamuliro wa zolozera zamkati: gwiritsani ntchito kaphatikizidwe koyambira/kafukufuku wa jini yachitsanzo kuti muyesedwe kuti muwone ngati template ingadziwike ndi PCR.

Zindikirani:

Zowongolera zabwino, zoyipa / zopanda kanthu komanso zowongolera zamkati ziyenera kukhazikitsidwa pamayeso aliwonse kuti muwone ngati zotsatira zoyeserera.

Kukonzekera kuyesa

Musanagwiritse ntchito, onani ngati yankho likusakanikirana mofanana.Ngati mvula ipezeka, iyenera kusungunuka ndikusakaniza molingana ndi malangizo musanagwiritse ntchito.Kusakaniza kwa 2 × PCR kumafunika kupakidwa ndi kusakaniza mobwerezabwereza ndi micropipette musanagwiritse ntchito kuti mupewe kugawanika kwa ion.

Zindikirani:

Tulutsani bukhuli ndikuliwerenga mosamala, ndipo konzekerani musanayambe kuyesa motsatira zofunikira za bukhuli.

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Konzani PCR reaction system

Malinga ndi protocol yoyeserera, sakanizani zoyambira, H2O, ndi 2 × PCR kusakaniza molingana, centrifuge ndikugawa ku chubu chilichonse chochitira.

Zindikirani:

Pakuyesa kwakukulu kapena kwanthawi yayitali, tikulimbikitsidwa kugwiritsa ntchito njira ya PCR yokhala ndi UNG enzyme, yomwe imatha kupewa kuipitsidwa ndi aerosol chifukwa cha zinthu za PCR.

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Onjezani zowonera

Pogwiritsa ntchito ukadaulo wa Direct PCR, sipafunikanso njira yotopetsa yoyeretsera ma nucleic acid, template yachitsanzo ikhoza kukonzedwa mkati mwa mphindi 10, ndipo njira yofananira ya PCR ikhoza kuwonjezeredwa.

Zindikirani:

Njira ya cleavage imakhala ndi zotsatira zabwino zodziwikiratu, ndipo zomwe mwapeza zitha kugwiritsidwa ntchito pozindikira zambiri.

5.1: Kukula kwachindunji kwa masamba

Malingana ndi kukula kwa chithunzi chomwe chili mu bukhuli, dulani minofu ya masamba ndi m'mimba mwake ya 2-3mm ndikuyiyika mu dongosolo la PCR.

Zindikirani: Onetsetsani kuti zidutswa zamasamba zamizidwa kwathunthu mu njira ya PCR reaction, ndipo musawonjezere masamba ochulukirapo.

5.2: Njira yogawa masamba

Dulani masamba a masamba ndi m'mimba mwake 5-7mm ndikuyika mu chubu cha centrifuge.Ngati mwasankha masamba okhwima, chonde pewani kugwiritsa ntchito minofu ya mtsempha waukulu wa tsamba.Pipette 50ul Buffer P1 lysate mu chubu cha centrifuge kuonetsetsa kuti lysate ikhoza kumiza minofu ya masamba, kuiyika mu cycler yotentha kapena kusamba kwachitsulo, ndi lyse pa 95 ° C kwa mphindi 5-10.

Onjezani 50ul Buffer P2 neutralization yankho ndikusakaniza bwino.The lysate chifukwa angagwiritsidwe ntchito ngati template ndi anawonjezera PCR reaction system.

Zindikirani: Kuchuluka kwa template kuli pakati pa 5-10% ya PCR system, ndipo zisapitirire 20% (mwachitsanzo, mu 20μl PCR system, kuwonjezera 1-2μl ya lysis solution, osapitirira 4μl).

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Zotsatira za PCR

Pambuyo centrifuging ndi PCR reaction chubu, ndi anaika mu PCR chida kuti matalikidwe.

Zindikirani:

Zomwe zimagwiritsa ntchito template yosayeretsedwa pakukulitsa, kotero kuchuluka kwa mizere yokulitsa ndi 5-10 yochulukirapo kuposa kugwiritsa ntchito template yoyeretsedwa ya DNA.

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Kuzindikira kwa Electrophoresis ndi kusanthula zotsatira

M: 100bp DNA Makwerero

1\4: Njira ya DNA yoyeretsedwa

2\5: Njira ya Direct PCR

3\6: Kuwongolera popanda kanthu

QC:

Zotsatira zoyeserera za maulamuliro osiyanasiyana omwe akhazikitsidwa pakuyesa ziyenera kukwaniritsa zotsatirazi.Apo ayi, chifukwa cha vutoli chiyenera kufufuzidwa, ndipo mayeserowo ayenera kuchitidwa kachiwiri vutolo litathetsedwa.

Table 1. Zotsatira zoyesa zamagulu osiyanasiyana olamulira

* plasmid ikagwiritsidwa ntchito ngati chiwongolero chabwino, zotsatira zoyeserera za majini zimatha kukhala zoyipa

Zotsatira zake:

A. Zotsatira za mayeso a jini yotsala yachitsanzoyo ndi yolakwika, kusonyeza kuti DNA yoyenera kuzindikiridwa wamba ya PCR siingatulutsidwe muchitsanzo kapena DNA yotengedwa ili ndi PCR reaction inhibitors, ndipo DNA iyenera kuchotsedwanso.

B. Zotsatira zoyesa za jini yokhazikika yachitsanzoyo ndi yabwino, ndipo zotsatira za jini yakunja ndi yolakwika, kusonyeza kuti DNA yoyenera kuzindikiridwa kwa PCR wamba imachotsedwa mu zitsanzo, ndipo zitha kuganiziridwa kuti jini ya XXX sinadziwike pachitsanzocho.

C. Zotsatira zoyesa za jini yokhazikika yachitsanzoyo ndi zabwino, ndipo zotsatira za jini yakunja ndizabwino, zomwe zikuwonetsa kuti DNA yoyenera kuzindikiridwa kwa PCR wamba yatulutsidwa muchitsanzo, ndipo DNA yachitsanzo ili ndi jini ya XXX.Mayesero otsimikizira akhoza kuchitidwanso.

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Zoyambira zozindikirira mapangidwe

Pambuyo poyesera, gwiritsani ntchito 2% sodium hypochlorite solution ndi 70% ethanol solution kuti mufufuze malo oyesera kuti muteteze kuipitsidwa kwa chilengedwe.