A matsayin sabon abu a cikin dakin gwaje-gwaje, ba aiki mai kyau ba ne don tantance shuke-shuke masu kyau daga gungun shuke-shuke tare da ƙarancin juyawa.Da fari dai, dole ne a fitar da DNA daga ɗimbin samfuri ɗaya bayan ɗaya, sannan PCR za ta gano kwayoyin halittar waje.Koyaya, sakamakon galibi ba komai bane da makada tare da wasu ƴan abubuwa lokaci-lokaci, amma ba zai yuwu a tantance ko akwai ganowar da aka rasa ko gano ƙarya ba..Shin rashin taimako ne sosai don fuskantar irin wannan tsari na gwaji da sakamako?Kada ku damu, ɗan'uwa yana koya muku yadda ake fitar da tsire-tsire masu kyau cikin sauƙi da daidai.

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Zane abubuwan ganowa

Ƙayyade ƙayyadaddun kwayoyin halitta da keɓaɓɓen kwayoyin halitta da za a gano bisa ga samfurin da za a gwada, kuma zaɓi wakilin 100-500bp a cikin kwayar halitta don ƙirar farko.Kyawawan firamare na iya tabbatar da daidaiton sakamakon ganowa kuma su rage lokacin ganowa (duba abin da aka ƙara don abubuwan gano abubuwan da aka saba amfani da su).

Sanarwa: Sabbin abubuwan da aka ƙera suna buƙatar haɓaka yanayin amsawa da tabbatar da daidaito, daidaito da iyakar ganowa kafin gano babban sikelin.

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Zane ƙa'idar gwaji

Kyakkyawan iko: Yi amfani da tsaftataccen DNA mai ɗauke da guntun manufa azaman samfuri don tantance ko tsarin amsawar PCR da yanayi na al'ada ne.

Ikon mara kyau/marasa: Yi amfani da samfurin DNA ko ddH2O wanda bai ƙunshi guntun manufa azaman samfuri don gano ko akwai tushen gurɓatawa a cikin tsarin PCR.

Ikon tunani na ciki: yi amfani da haɗe-haɗe na farko/bincike na ƙayyadaddun kwayoyin halittar samfurin da za a gwada don kimanta ko za a iya gano samfurin ta PCR.

Sanarwa:

Ya kamata a saita madaidaicin iko, mara kyau / mara kyau da sarrafawa na ciki don kowane gwaji don kimanta ingancin sakamakon gwaji.

Gwaji shiri

Kafin amfani, lura ko an haɗa maganin daidai gwargwado.Idan an sami hazo, yana buƙatar narkar da shi kuma a gauraye shi bisa ga umarnin kafin amfani.Haɗin 2 × PCR yana buƙatar bututu kuma a gauraya akai-akai tare da micropipette kafin amfani da shi don guje wa rarraba ion mara kyau.

Sanarwa:

Fitar da littafin kuma karanta shi a hankali, kuma kuyi shirye-shirye kafin gwaji daidai da buƙatun littafin.

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Shirya tsarin amsawar PCR

Dangane da ka'idar gwaji, haɗa abubuwan farko, H2O, da 2 × PCR suna haɗuwa daidai, centrifuge kuma rarraba su zuwa kowane bututu mai amsawa.

Sanarwa:

Don gwaji mai girma ko na dogon lokaci, ana ba da shawarar yin amfani da tsarin amsawar PCR mai ɗauke da enzyme UNG, wanda zai iya guje wa gurɓacewar iska ta hanyar samfuran PCR.

Mataki na 5

Ƙara samfurin amsawa

Yin amfani da fasahar PCR kai tsaye, babu buƙatar aiwatar da aikin tsarkakewa na nucleic acid mai wahala, ana iya shirya samfurin samfurin a cikin mintuna 10, kuma ana iya ƙara tsarin amsawar PCR daidai.

Sanarwa:

Hanyar tarwatsewa tana da mafi kyawun tasirin ganowa, kuma ana iya amfani da samfurin da aka samu don halayen ganowa da yawa.

5.1: Kai tsaye fadada ganye

Dangane da girman hoton da ke cikin littafin, yanke nama na ganye tare da diamita na 2-3mm kuma sanya shi a cikin tsarin amsawar PCR.

Lura: Tabbatar cewa gutsuttsuran ganyen sun nutse gaba ɗaya a cikin maganin amsawar PCR, kuma kar a ƙara naman ganyen da ya wuce kima.

5.2: Hanyar raba ganye

Yanke naman ganye tare da diamita na 5-7mm kuma sanya shi a cikin bututun centrifuge.Idan ka zaɓi manyan ganye, da fatan za a guji amfani da kyallen jikin babban jijiyar ganyen.Pipette 50ul Buffer P1 lysate a cikin bututu na centrifuge don tabbatar da cewa lysate na iya nutsar da nama na ganye gaba ɗaya, sanya shi a cikin injin hawan zafi ko wanka na ƙarfe, da lyse a 95 ° C na minti 5-10.

Ƙara 50ul Buffer P2 maganin neutralization kuma haxa da kyau.Sakamakon lysate za a iya amfani dashi azaman samfuri kuma ƙara zuwa tsarin amsawar PCR.

Lura: Adadin samfuri yana tsakanin 5-10% na tsarin PCR, kuma kada ya wuce 20% (misali, a cikin tsarin 20μl PCR, ƙara 1-2μl na maganin lysis, bai wuce 4μl).

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Amsar PCR

Bayan centrifuging na PCR dauki bututu, an sanya shi a cikin na'urar PCR don haɓakawa.

Sanarwa:

Halin yana amfani da samfurin da ba a tsarkake ba don haɓakawa, don haka adadin zagayowar haɓakawa shine ƙarin hawan keke 5-10 fiye da lokacin amfani da samfurin DNA mai tsafta.

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Binciken Electrophoresis da bincike na sakamako

M: Tsani na DNA 100bp

1\4: Tsaftataccen hanyar DNA

2\5: Hanyar PCR kai tsaye

3\6: Gudanar da komai

QC:

Sakamakon gwajin nau'ikan sarrafawa daban-daban da aka saita a cikin gwajin yakamata ya dace da waɗannan sharuɗɗan.In ba haka ba, sai a yi nazari kan musabbabin matsalar, sannan a sake yin gwajin bayan an kawar da matsalar.

Tebur 1. Sakamakon gwajin al'ada na ƙungiyoyin sarrafawa daban-daban

*Lokacin da aka yi amfani da plasmid a matsayin ingantaccen iko, sakamakon gwajin kwayoyin halitta na iya zama mara kyau

Hukuncin sakamako:

A. Sakamakon gwajin kwayar halittar endogenous na samfurin ba shi da kyau, yana nuna cewa DNA ɗin da ta dace da gano PCR na yau da kullun ba za a iya fitar da shi daga samfurin ba ko kuma DNA ɗin da aka ciro ya ƙunshi masu hana amsawar PCR, kuma ya kamata a sake fitar da DNA.

B. Sakamakon gwajin kwayoyin halitta na samfurin yana da kyau, kuma sakamakon gwajin kwayoyin halitta ba shi da kyau, yana nuna cewa DNA wanda ya dace da ganewar PCR na yau da kullum an fitar da shi daga samfurin, kuma ana iya yanke hukunci cewa ba a gano kwayoyin XXX a cikin samfurin ba.

C. Sakamakon gwajin kwayar halittar endogenous na samfurin yana da kyau, kuma sakamakon gwajin da aka yi na exogenous gene yana da kyau, yana nuna cewa an fitar da DNA da ya dace da gano PCR na yau da kullun daga samfurin, kuma samfurin DNA ya ƙunshi kwayar halittar XXX.Ana iya ƙara yin gwajin tabbatarwa.

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Zane abubuwan ganowa

Bayan gwajin, yi amfani da 2% sodium hypochlorite bayani da 70% ethanol bayani don shafe yankin gwaji don hana gurɓataccen muhalli.