Dubawa
Saurin gano tsire-tsire na transgenic
Rubutu/Tong Yucheng
Ayyukan gwaji/Han Ying
Edita/Wen Youjun
Kalmomi/1600+
Shawarwari lokacin karantawa/minti 8-10
Saurin gano tsire-tsire na transgenic
A matsayin sabon shiga 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.
Mataki na 1: 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).
Lura:
Sabbin gyare-gyaren da aka ƙera suna buƙatar haɓaka yanayin amsawa da tabbatar da daidaito, daidaito, da iyakar ganowa kafin aiwatar da gano babban sikelin.
Mataki na 2:Ƙirƙirar 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/rabo: Yi amfani da samfurin DNA ko ddH2O wanda bai ƙunshi guntun manufa azaman samfuri don gano ko akwai tushen gurɓatawa a cikin tsarin PCR ba.
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.
Lura:
Ya kamata a saita madaidaicin iko, mara kyau / mara kyau da sarrafawa na ciki don kowane gwaji don kimanta ingancin sakamakon gwaji.
Mataki na 3: 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.
Lura:
Fitar da umarnin kuma karanta su a hankali, kuma ku yi shirye-shirye kafin gwajin daidai da umarnin.
Mataki 4: Shirya tsarin amsawar PCR
Bisa ga ka'idar gwaji, haɗa abubuwan da aka tsara, H2O, 2 × PCR Mix, centrifuge kuma rarraba su zuwa kowane bututu mai amsawa.
Lura:
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 5: Ƙara samfurin amsawa
Amfani da fasahar PCR kai tsaye, babu buƙatar aiwatar da tsaftar acid nucleic mai wahala.Za'a iya shirya samfurin samfurin a cikin mintuna 10 kuma ƙara zuwa tsarin amsa PCR daidai.
Lura:
Hanyar Lysis tana da sakamako mafi kyawun ganowa, kuma ana iya amfani da samfurin da aka samu don halayen ganowa da yawa.
5.1: PCR kai tsaye na 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 lysis leaf
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 yakamata ya kasance tsakanin 5-10% na tsarin PCR, kuma kada ya wuce 20% (misali, a cikin tsarin 20μl PCR, ƙara 1-2μl na buffer lysis, bai wuce 4μl).
Mataki 6: Amsa PCR
Bayan centrifuging na PCR dauki bututu, sanya su a cikin PCR kayan aiki don ƙarawa.
Lura:
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.
Mataki na 7: Gano Electrophoresis da bincike na sakamako
M: 100bp DNA Tsani
1\4: Tsaftataccen hanyar DNA
2\5: Hanyar PCR kai tsaye
3\6: Gudanar da komai
Kula da inganci:
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 kwayar 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 ɗin da ya dace don gano PCR na yau da kullun daga samfurin, kuma samfurin DNA yana ɗauke da kwayar halittar XXX.Ana iya ƙara yin gwajin tabbatarwa.
Mataki 8: Ƙirar gano abubuwan ƙira
Bayan gwajin, yi amfani da maganin 2% sodium hypochlorite da 70% maganin ethanol don shafe yankin gwaji don hana gurɓataccen muhalli.
Karin bayani
Tebur 2. Abubuwan da aka saba amfani da su don gano PCR na gabaɗaya na tsire-tsire da aka gyara
Takardun Magana:
SN/T 1202-2010, Ingantacciyar hanyar gano PCR don abubuwan shuka da aka gyara a cikin abinci.
Sanarwa na Ma'aikatar Aikin Noma 1485-5-2010, Gwajin sinadaran da aka gyara ta hanyar kwayoyin halitta da kayayyakinsu- Shinkafa M12 da abubuwan da suka samo asali.
Lokacin aikawa: Juni-09-2021
