銆愮敘(ch菐n)鍝佺法铏�(h脿o)銆�2793950
銆愯嫳鏂囧悕绋便��Locus-specific, quantitative 5fC analysis Kit
銆愪腑鏂囧悕绋便��鐗瑰畾浣嶉粸(di菐n)5-閱涘熀鑳炲槯鍟跺畾閲忔娓�(c猫)瑭﹀姂鐩�
銆愮敘(ch菐n)鍝佸収(n猫i)瀹广��
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鐢�(ch菐n)鍝佺祫鎴�
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鍚噺
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婧舵恫L1锛�Buffer L1锛�
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2.4 mL
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M
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24绠�
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妯″紡搴忓垪
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24ul
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娉細婧舵恫L1锛氫笝浜岃厛鍙嶆噳(y墨ng)娑诧紱M锛氫笝浜岃厛
銆愪繚瀛樻浠躲��妯″紡搴忓垪缃簬-20 °C锛屽彲淇濆瓨12鍊�(g猫)鏈堛��
M鍜屾憾娑�L1缃簬4 °C锛屽彲淇濆瓨12鍊�(g猫)鏈�銆�
M闇�閬垮厜淇濆瓨銆�
銆愮敘(ch菐n)鍝佹杩般��
5-鐢插熀鑳炲槯鍟朵慨椋撅紙5mC锛夋槸鐪熸牳绱�(x矛)鑳炲熀鍥犵祫涓渶鐐洪噸瑕佺殑琛ㄨ閬哄偝淇＞涔嬩竴銆�5mC鍙互鍦�TET瀹舵棌閰剁殑浣滅敤涓嬮�愭姘у寲鐢熸垚5-缇ョ敳鍩鸿優鍢у暥锛�5hmC锛�銆�5-閱涘熀鑳炲槯鍟讹紙5fC锛夊拰5-缇у熀鑳炲槯鍟讹紙5caC锛�锛涘皪(du矛)浜庝笂杩板咕绋柊鍨�DNA淇＞鐨勫姛鑳界爺绌舵槸鐩墠鏍搁吀琛ㄨ閬哄偝瀛�(xu茅)鐨勪竴鍊�(g猫)鐮旂┒鐔遍粸(di菐n)銆�
鐧鹃潏濞佹帹鍑烘柊鍨�5fC妾㈡脯(c猫)瑭﹀姂鐩�锛屽畠涓昏鐢ㄤ簬5-閱涘熀鑳炲槯鍟讹紙5-formylcytosine, 5fC锛夌殑鍏ㄥ熀鍥犵祫鍠牽鍩哄垎杈ㄧ巼娓�(c猫)搴�锛涗篃鍙噳(y墨ng)鐢ㄤ簬5fC鍦ㄧ敓鐗╃櫦(f膩)鑲插拰鐤剧梾鐧�(f膩)鐢熺櫦(f膩)灞曢亷绋嬬殑鐢熺墿瀛�(xu茅)鍔熻兘鐮旂┒銆�
銆愯嚜鍌欐潗鏂欍��
• Oligo Clean & Concentrator (ZYMO, #D4060)
• MightyAmp® DNA Polymerase Ver.3(TaKaRa, Cat.#R076A)
• 鐒℃按涔欓唶
• 婊呰弻瓒呯磾姘�
• 浣庡惛闄�EP銆�PCR绠�
• PCR鐔卞惊鐠�(hu谩n)鍎�
• Thermo-mixer (Eppendorf)
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A.閬垮厤妯ｅ搧浜ゅ弶姹℃煋
鍚稿彇涓嶅悓妯ｅ搧鏅�(sh铆)鏇存彌妲嶉牠
B.閰剁殑鍙栨斁鎳�(y墨ng)鍦ㄥ啺涓婇��(j矛n)琛岋紝鐐洪伩鍏嶅弽寰�(f霉)鍑嶈瀺鎴栭暦(zh菐ng)鏈熶娇鐢ㄥ悗娲绘�т笅闄�,鎴戝�戞帹钖︽偍鍦ㄩ娆′娇鐢ㄥ悗灏囧墿浣欒│鍔戝皬浠藉垎瑁濆噸瀛�
銆愬(sh铆)椹�(y脿n)姝ラ銆�
璧峰鏉愭枡锛氱磾鍖栧悗鐨勫熀鍥犵祫DNA锛屾憾浜�EB涓�銆�
DNA绱斿害瑕佹眰锛�OD260锛�OD280锛�1.8锝�2.0
1锛夊湪绱斿寲鐨勫熀鍥犵祫DNA涓姞鍏�1ul M锛�1ul婧舵恫L1鍜�1ul妯″紡搴忓垪锛屽厖鍒嗘贩鍕�锛屽皣鍏剁疆浜�Thermo-mixer (Eppendorf)涓�锛�37°C锛�850rmp閬垮厜鍙嶆噳(y墨ng)20h銆�
2锛�5fC妯�(bi膩o)瑷樺畬鎴愬悗锛屾寜鐓�MightyAmp® DNA Polymerase Ver.3(TaKaRa, Cat.#R076A)瑾槑鏇稿皪(du矛)鐗圭暟鎬х殑浣嶉粸(di菐n)閫�(j矛n)琛�PCR鎿�(ku貌)澧炪��
3锛夋寜鐓�Oligo Clean & Concentrator (ZYMO, #D4060)瑾槑鏇哥磾鍖�PCR鍚庣殑DNA, 鐢�30ul 瓒呯磾姘存礂鑴�锛屼簬-20°C淇濆瓨銆�